Methods for propagating adenovirus and virus produced thereby

ABSTRACT

Various methods for propagating and rescuing multiple serotypes of replication-defective adenovirus in a single adenoviral E1-complementing cell line are disclosed. Typically, replication-defective adenovirus vectors propagate only in cell lines which express E1 proteins of the same serotype or subgroup as the vector. The disclosed methods offer the ability to propagate vectors derived from multiple adenoviral serotypes in a single production cell line which expresses E1 proteins from a single serotype. Propagation in this manner is accomplished by providing all or a portion of an E4 region in cis within the genome of the replication-defective adenovirus. The added E4 region or portion thereof is cloned from a virus of the same or highly similar serotype as that of the E1 gene product(s) of the complementing cell line. Interaction between the expressed E1 of the cell line and the heterologous E4 of the replication-defective adenoviral vectors enables their propagation and rescue. The invention bypasses a need in the art to customize specific cell lines to the serotype or subgroup of the adenoviral vector being propagated and enables one to easily and rapidly develop alternative adenoviral serotypes as gene delivery vectors for use as vaccines or as a critical component in gene therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of application serial No. 60/458,825, filed Mar. 28, 2003; No. 60/455,312, filed Mar. 17, 2003; No. 60/455,234, filed Mar. 17, 2003; and No. 60/405,182, filed Aug. 22, 2002.

FIELD OF THE INVENTION

[0002] The present invention concerns various methods to propagate and rescue multiple serotypes of replication-defective adenovirus in a single adenoviral E1-complementing cell line. Typically, replication-defective adenovirus vectors propagate only in cell lines which express E1 proteins of the same serotype or subgroup as the vector. The methods disclosed herein offer the ability to propagate vectors derived from multiple serotypes in a single cell line expressing E1 proteins from a single serotype. Such propagation of a wide range of vectors in one cell line is accomplished by providing all or a portion of an E4 region in cis within the genome of the replication-defective adenovirus. The added E4 region or portion thereof is cloned from a virus of the same or highly similar serotype as that of the E1 gene product(s) of the complementing cell line. Interaction between the E1 gene products of the cell line and the heterologous E4 gene products of the replication-defective adenoviral vector enables the propagation and rescue of the recombinant replication-defective adenovirus vectors. The invention, therefore, bypasses an existing need in the art to customize complementing cell lines to the specific serotype or subgroup of the adenoviral vector being propagated or, alternatively, to have to transfect a cell line with an E4 region and then regulate the expression in trans of the E4 region within the E1 complementing cell line.

BACKGROUND OF THE INVENTION

[0003] Beginning with the first human adenoviruses (Ads) isolated over four decades ago (Rowe et al., Proc. Soc. Exp. Biol. Med., 84:570-579, 1953), over 100 distinct serotypes of adenovirus have been isolated which infect various mammalian species, 51 of which are of human origin (Straus, Adenovirus infections in humans. In The Adenoviruses. 451-498, 1984; Hierholzer et al., J. Infect. Dis., 158: 804-813, 1988; Schnurr and Dondero, Intervirology., 36: 79-83, 1993; Jong et al., J Clin Microbiol., 37:3940-3945:1999). The human serotypes have been categorised into six subgenera (A-F) based on a number of biological, chemical, immunological and structural criteria; criteria which include hemagglutination properties of rat and rhesus monkey erythrocytes, DNA homology, restriction enzyme cleavage patterns, percentage of G+C content and oncogenicity (Straus, Adenovirus infections in humans. In The Adenoviruses. 451-498, 1984; Horwitz, Adenoviridae and their replication, In Virology: 1679-172, 1990).

[0004] Deletion of an essential E1 region common to the various adenovirus serotypes has enabled the use of adenovirus vectors as gene transfer vectors for vaccine and gene therapy purposes. Resultant replication-defective vectors are propagated in cell lines that provide the deleted E1 gene products in trans. Supplementation of the essential E1 gene products in trans in this manner works well when the E1 gene products are from the same or a highly similar serotype. As such, E1-deleted group C serotypes (Ad1, Ad2, Ad5 and Ad6) grow well in 293 or PER.C6 cells which contain and express the Ad5 E1 region. In contrast, E1-deleted serotypes other than group C, for example those from subgroups A, B, D, E, and F (e.g., Ad3, Ad4, and Ad7 to Ad51), do not replicate efficiently in 293 or PER.C6 cells. The Ad5 E1 sequences in 293 and PER.C6 cells do not fully complement the replication of these alternative serotypes. This presents a challenge due to the fact that the most characterized and studied complementing cell lines available for growth and propagation of adenovirus are based on E1 sequence from adenovirus serotype 5.

[0005] This inability to fully complement the replication of serotypes other than group C adenovirus in Ad15 E1 complementing cell lines has been attributed to the inability of Ad5 (group C) E1b 55K gene product to functionally interact with the E4 gene products of non-group C serotypes. While the interaction is conserved within members of the same subgroup, it is not well conserved between subgroups.

[0006] Hence, cell lines expressing both Ad5 E1 and ORF6 were generated and proved useful in complementing alternative adenovirus serotypes; see, e.g., Abrahamsen et al., 1997 J. Virol. 8946-8951. Such incorporation of E4 (or ORF6) into Ad 5 complementing cell lines as was done in Abrahamsen et al., supra, is known.

[0007] U.S. Pat. No. 5,849,561 discloses complementation of an E1-deleted non-group C adenovirus vector in an Ad5-E1 complementing cell line which also expresses portions of the Ad5-E4 gene.

[0008] U.S. Pat. No. 6,127,175, issued to Vigne, et al., discloses a stably transfected mammalian cell line which expresses a portion of the E4 region of adenovirus, preferably ORF6 or ORF6/7. Such a cell line is useful for complementation of recombinant Ad genomes deficient in the E4 region.

[0009] European Application EP 1 054 064 A1 discloses recombinant, replication deficient adenovirus 35 (Ad35) vectors and cell lines which complement in trans the growth of these vectors. A cell line which expresses Ad5E1A and E2A genes (PER.C6) was shown to complement an Ad35-E1 deleted vector upon co-expression of Ad35-E1B proteins.

[0010] U.S. Pat. No. 6,270,996, issued to Wilson, et al., discloses E1/E4 deleted adenovirus vectors and E1/E4 (ORF6) cell lines which complement in trans virus growth without resulting in cell toxicity.

[0011] U.S. Pat. No. 6,202,060, issued to Mehtali, et al., discloses adenoviral vectors wherein portions of the early genes are under control of an inducible promoter. The '060 patent also discloses complementing cell lines which may be used in tandem with these Ad vectors.

[0012] The generation of serotype-specific cell lines providing a complementing serotype-specific E1 gene product(s) in trans is known as well.

[0013] Although Ad5-based vectors have been used extensively in a number of gene therapy trials, there may be limitations on the use of Ad5 and other group C adenoviral vectors due to preexisting immunity in the general population due to natural infection. Ad5 and other group C members tend to be among the most seroprevalent serotypes. Immunity to existing vectors may develop as a result of exposure to the vector during treatment. These types of preexisting or developed immunity to seroprevalent gene delivery vectors may limit the effectiveness of gene therapy or vaccination efforts. Alternative adenovirus serotypes, thus, constitute very important targets in the pursuit of gene delivery systems capable of evading the host immune response.

[0014] There remains both a practical and commercial need for an adenovirus-based vaccine and/or gene therapy delivery system which allows for the production of multiple serotype recombinant adenovirus vectors in a single source complementing mammalian cell line. The present invention addresses and overcomes this deficiency in the art by disclosing novel methods for propagating multiple serotype recombinant Ad vectors in a single complementing cell line where the required serotype-specific sequences are provided in cis.

SUMMARY OF THE INVENTION

[0015] The present invention relates to an enhanced means for propagating replication-defective adenovirus in an E1-complementing cell line(s) where the E1 gene product(s) being expressed is not native to the adenovirus being propagated. The method is based on Applicants' finding that supply, in cis, of a nucleic acid sequence encoding all or a portion of a heterologous adenoviral E4 region which is native to a virus of the same or highly similar serotype as the E1 gene product(s) of the complementing cell line enables the growth of adenoviral vectors of varying serotype in any single complementing cell line, despite the fact the cell line is not customized for the particular serotype of vector being propagated. This is of particular importance given that existing and settled adenoviral E1-complementing cell lines (such as PER.C6™ and 293) are based on one of the most prominent adenovirus serotypes (Ad5) and are not suited for the large-scale propagation and rescue of alternative serotypes.

[0016] The basic steps involved in the propagation of adenoviral vectors in accordance with the methods of the instant invention are as follows: First, all or a portion of a heterologous adenoviral E4 region comprising nucleic acid sequence encoding at least open reading frame 6 (ORF6) is inserted into a replication-defective adenoviral vector. By “heterologous”, Applicants mean that the nucleic acid sequence is not native to the viral vector being propagated, i.e., not normally present within a virus of the same or highly similar serotype. As will be described, the adenoviral E4 region or portion thereof can be either a nucleic acid sequence encoding ORF 6 or any larger portion of the E4 region, and includes nucleic acid comprising the complete E4 region with E4 promoter. The region into which the nucleic acid is incorporated is not limited, i.e., the insertion can be made into the complete E4 region with E4 promoter or into a smaller portion narrowing into the ORF6 region. Alternatively, the heterologous E4 region or portion thereof can be inserted into different areas of the genome such as the E1 or E3 regions. Further, the native E4 region or portion thereof can be deleted and replaced, or left intact. This is not deemed a critical element of the instant invention. What is a critical element is that the heterologous E4 region or portion thereof being inserted is native to a virus of the same or highly similar serotype as the E1 gene product(s) expressed by the complementing cell line.

[0017] Following the modification of the adenoviral vector of interest, the recombinant adenovirus is then introduced into an adenoviral E1-complementing cell line and allowed to propagate. The adenovirus is subsequently harvested and rescued from the complementing cell line.

[0018] The resultant virus can be studied and used in various gene therapy and vaccine efforts. The virus, therefore, forms an important aspect of the instant invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 illustrates a transcription map for adenovirus serotype 5. The linear genome is divided into 100 map units as well as into r- and l-strands which designate the direction of transcription. Early transcription units are designated with an E and are active prior to viral DNA replication. Late transcription units are designated with and L and are active primarily after DNA replication. Promoters are represented as brackets and polyadenylation sites as arrowheads. The tripartite leader is designated 1, 2, and 3.

[0020] FIGS. 2A-1 through 2A-10 illustrate the nucleic acid sequence for the wild-type adenovirus 35 (SEQ ID NO: 1) utilized in the Examples.

[0021]FIG. 3 illustrates the homologous recombination scheme utilized to recover pAd35ΔE1.

[0022]FIG. 4 illustrates the various configurations of the E4 regions (or portions) within the alternative serotype recombinants.

[0023]FIG. 5 illustrates the homologous recombination scheme utilized to recover pAd35ΔE1ΔE4Ad5Orf6.

[0024]FIG. 6 illustrates the nucleic acid sequence encoding the gag expression cassette (SEQ ID NO: 2). The various regions of the figure are as follows: (1) a first underlined segment of nucleic acid sequence encoding the immediate early gene promoter region from human cytomegalovirus; (2) a first segment of lowercase letters which is not underlined, which segment of DNA contains a convenient restriction enzyme site; (3) a region in caps which contains the coding sequence of HIV-1 gag; (4) a second segment of lowercase letters which is not underlined, which segment of DNA contains a convenient restriction enzyme site; and (5) a second underlined segment, this segment containing nucleic acid sequence encoding a bovine growth hormone polyadenylation signal sequence.

[0025]FIG. 7 illustrates the nucleic acid sequence encoding the SEAP expression cassette (SEQ ID NO: 3). The various regions of the figure are as follows: (1) a first underlined segment of nucleic acid sequence encoding the immediate early gene promoter region from human cytomegalovirus; (2) a first segment of lowercase letters which is not underlined, which segment of DNA contains a convenient restriction enzyme site; (3) a region in caps which contains the coding sequence of the human placental SEAP gene; (4) a second segment of lowercase letters which is not underlined, which segment of DNA contains a convenient restriction enzyme site; and (5) a second underlined segment, this segment containing nucleic acid sequence encoding a bovine growth hormone polyadenylation signal sequence.

[0026]FIG. 8 illustrates in vivo expression of SEAP in C3H/HeN mice using 10{circumflex over ( )}10 vp doses of Ad35 vectors. This experiment was designed to address any effects of E3 deletion. The vectors were injected intramuscularly and the levels of SEAP expression were determined from the serum samples. Shown are geometric means for each cohort of 5 mice.

[0027]FIG. 9 illustrates in vivo expression of SEAP in C3H/HeN mice using 10{circumflex over ( )}10 vp doses of Ad35 vectors. This experiment was designed to address any effects of Ad5 sequence insertion into the Ad35 genome. The vectors were injected intramuscularly and the levels of SEAP expression were determined from the serum samples. Two extra cohorts received 10{circumflex over ( )}10 vp and 10{circumflex over ( )}9 vp of Ad5 vector. Shown are geometric means for each cohort of 5 mice.

[0028] FIGS. 10A-B illustrate in vivo SEAP expression using MRKAd5-based (A) and Ad35ΔE1ΔE4Ad5Orf6-based (B) vector in rhesus macaques. Shown are the serum antigen levels for individual monkeys following a single intramuscular (i.m.) injection of 10{circumflex over ( )}11 vp MRKAd5SEAP (filled circles), 10{circumflex over ( )}9 vp MRKAd5SEAP (open boxes) or 10{circumflex over ( )}11 vp Ad35ΔE1SEAPΔE4Ad5Orf6.

[0029]FIG. 11 illustrates in vivo SEAP expression in African green monkeys using Ad5- and Ad35-based vectors. Shown are the antigen levels for each animal in serum samples collected two days after the treatment.

[0030]FIG. 12 illustrates the homologous recombination scheme utilized to recover pAd24ΔE1.

[0031]FIG. 13 illustrates the homologous recombination scheme utilized to recover pAd24ΔE1Ad5Orf6.

[0032]FIG. 14 illustrates the configuration of E4 regions in the Ad24 recombinants generated.

[0033]FIG. 15 illustrates the growth kinetics of the Ad24-based vectors in PER.C6 cells.

[0034] FIGS. 16A-1 through 16A-10 illustrate the nucleic acid sequence for wild-type adenovirus serotype 24 (SEQ ID NO: 5). The ATCC product number for Ad24 is VR-259.

[0035]FIG. 17 illustrates, in tabular format, gag-specific T cell responses in monkeys immunized with MRKAd5-HIVgag and Ad24 HIV vectors. Shown are the numbers of spot-forming cells per million PBMC following incubation in the absence (mock) or presence of Gag peptide pool. The pool consisted of 20-aa peptide overlapping by 10 aa and encompassing the entire gag sequence.

[0036]FIG. 18 illustrates, in tabular format, the characterization of the gag-specific T cells in monkeys immunized with 10{circumflex over ( )}11 vp of MRKAd5-HIV1gag and Ad24ΔE1gagΔOrf6Ad5Orf6. Shown are the percentages of CD3+ T cells that are either gag-specific CD4+ or gag-specific CD8+ cells. These values were corrected for mock values (<0.03%).

[0037]FIG. 19 illustrates individual anti-p24 titers (in mMU/mL) in macaques immunized with gag-expressing adenovirus vectors.

[0038]FIG. 20 illustrates in vivo expression of SEAP in C3H/HeN mice using 10{circumflex over ( )}10 vp doses of Ad24 vectors. The vectors were injected intramuscularly and the levels of SEAP expression were determined from the serum samples. Two extra cohorts received 10{circumflex over ( )}10 vp and 10{circumflex over ( )}9 vp of Ad5 vector. Shown are geometric means for each cohort of 5 mice.

[0039]FIG. 21 illustrates in vivo SEAP expression using MRKAd5 and Ad24 vectors in rhesus macaques. Shown are the geometric means of the SEAP levels for cohorts of 3 monkeys. In bars are the standard errors of the geometric means.

[0040]FIG. 22 illustrates a homologous recombination scheme to be utilized to recover pAd24ΔE1ΔE4Ad5Orf6.

[0041]FIG. 23 illustrates gag-specific T cell responses in rhesus macaques immunized following a heterologous Ad5/Ad6 prime-Ad24 boost regimen. a: Mock, no peptide: gag, 20-mer peptide pool encompassing entire gag sequence; b: Peak response after 2 or 3 doses of the priming vaccine; c: 3 wks prior to boost; d: 4 wks after boost; e: ND, not determined.

[0042]FIG. 24 illustrates, in tabular format, the percentages of CD3⁺ T lymphocytes that are gag-specific CD8⁺ cells or gag-specific CD4⁺ cells determined after the Ad24 Boost Immunization (wk 60). Numbers reflect the percentages of circulating CD3+ lymphocytes that are either gag-specific CD4+ or gag-specific CD8+ cells. Mock values (equal to or less than 0.01%) have been subtracted.

[0043]FIG. 25 illustrates gag-specific T cell responses in rhesus macaques immunized following a heterologous Ad 24 prime-Ad5 boost regimen. a: Mock, no peptide: gag, 20-mer peptide pool encompassing entire gag sequence; b: Peak response after 2 doses of the priming vaccine; c: Wk 24; d: 4 wks after boost; e: ND, not determined.

[0044]FIG. 26 illustrates the homologous recombination scheme utilized to recover pAd34ΔE1ΔE4Ad5Orf6.

[0045]FIG. 27 illustrates the homologous recombination scheme utilized to recover pMRKAd34ΔE1ΔE4Ad5Orf6.

[0046] FIGS. 28A-1 to 28A-9 illustrate a nucleic acid sequence for wild-type adenovirus serotype 34 (SEQ ID NO: 12). The ATCC product number for Ad34 is VR-716.

[0047]FIG. 29 illustrates the time course of SEAP expression using MRKAd5 and Ad34 vectors in rhesus macaques. Data represent cohort geometric means.

[0048]FIG. 30 illustrates, in tabular format, T cell responses induced using MRKAd5 and Ad34 vectors expressing HIV-1 gag. Data are expressed in numbers of spot-forming cells per million PBMC (SFC/10{circumflex over ( )}6 PBMC). “a” refers to a 20-mer peptide pool with 10-aa overlap and encompassing the entire HIV-1 CAM1 gag.

[0049]FIG. 31 illustrates, in tabular format, the levels of CD4+ and CD8+ Gag-specific T cells in Ad34-immunized macaques at week 12. “a” refers to a 20-mer peptide pool with 10-aa overlap and encompassing the entire HIV-1 CAM1 gag.

[0050]FIG. 32 illustrates, in tabular format, T cell responses induced using a heterologous Ad34 prime/Ad35 boost regimen in macaques. “a” refers to a 20-mer peptide pool with 10-aa overlap and encompassing the entire HIV-1 CAM1 gag.

[0051]FIG. 33 illustrates, in tabular format, the levels of CD4+ and CD8+ Gag-specific T cells in Ad34 primed/Ad35 boosted macaques at week 28. “a” refers to a 20-mer peptide pool with 10-aa overlap and encompassing the entire HIV-1 CAM1 gag.

DETAILED DESCRIPTION OF THE INVENTION

[0052] The present invention details an efficient strategy for the propagation and rescue of alternative adenoviral serotypes utilizing available adenovirus production cell lines, nullifying the need to customize available cell lines for a specific serotype of interest. This is enabled by the incorporation of a critical E4 region into the adenovirus to be propagated.

[0053] The critical E4 region in the instant invention comprises, in the minimum, nucleic acid sequence encoding E4 ORF6 and can comprise the entire region of E4, inclusive of the promoter region. An important characteristic of the imported E4 region is that it is native to a virus of the same or highly similar serotype as the E1 gene product(s) (particularly E1B 55K) of the E1-complementing cell line, but heterologous to (i.e., non-native to a virus of the same serotype as) the adenoviral vector being propagated. As will be detailed below, the heterologous E4 region or portion thereof can be varied and can be inserted into the vector backbone at numerous locations.

[0054] The heterologous E4 region or portion thereof can, for instance, be a nucleic acid sequence encoding the entire open reading frame of the non-native E4. This segment of nucleic acid sequence can, in turn, be incorporated into the “native” entire E4 open reading frame of the recipient virus. In such an embodiment, the promoter native to the adenoviral vector would drive the expression of the non-native E4 region within the recombinant replication-defective adenoviral vector. Alternatively, the nucleic acid sequence encoding the entire open reading frame can be inserted into a different region of the adenoviral vector genome, such as for example the E1 or E3 regions. In this latter embodiment, the native E4 region or portion thereof can be deleted or left intact.

[0055] In another embodiment, the heterologous E4 region comprises a nucleic acid sequence encoding the entire open reading frame of E4 and includes a non-native E4 promoter. In this type of embodiment, the E4 region can be inserted into the location of the combined native E4 and E4 promoter region. The non-native E4 region in this embodiment would be driven by expression of the non-native E4 promoter. Alternatively, the nucleic acid sequence encoding the entire open reading frame and the non-native E4 promoter can be inserted into a different region of the adenoviral vector genome, such as for example the E1 or E3 regions. In this latter embodiment, the native E4 region or portion thereof can be deleted or left intact.

[0056] An alternative and further embodiment exists wherein the heterologous E4 region or portion thereof comprises nucleic acid sequence encoding a partial E4 region comprising ORF6 (one aspect of which is a region solely encoding ORF6). In this particular aspect of the invention, the heterologous non-native E4 protein can, in certain embodiments, replace the nonnative ORF6 region or the entire E4-encoding region of the native virus. In the latter situation, the promoter driving expression of the non-native ORF6 can either be the native E4 promoter or a heterologous, non-native promoter operatively linked to the non-native ORF6, while in the latter, the expression of the non-native ORF6 would generally be driven by the native E4 promoter. Alternatively, the nucleic acid sequence encoding a partial E4 region comprising ORF 6 can be inserted into a different region of the adenoviral vector genome, such as for example the E1 or E3 regions. In this latter embodiment, the native E4 region or portion thereof can be deleted or left intact.

[0057] As one of skill in the art can appreciate, there are various ways in which one can envision the supply of a heterologous E4 nucleic acid sequence in cis to an adenoviral vector and thereby enable its growth based on Applicants' novel findings herein. Moreover, as one of skill in the art can appreciate, either native or non-native promoters can be utilized to drive expression of the heterologous E4 region or portion thereof.

[0058] Adenovirus pre-plasmids (plasmids comprising the genome of the replication-defective adenovirus with desired deletions and insertions) can be generated by homologous recombination using adenovirus backbones and an appropriate shuttle vector (designed to target-in specific deletions and incorporate desired restriction sites into the resultant plasmid). Shuttle vectors of use in this process can be generated using general methods widely understood and appreciated in the art, e.g., PCR of the adenoviral terminal ends taking into account the desired deletions, and the sequential cloning of the respective segments into an appropriate cloning plasmid. The adenoviral pre-plasmid can then be digested and transfected into the complementing cell line via calcium phosphate co-precipitation or other suitable means. Virus replication and amplification then occurs, a phenomenon made evident by notable cytopathic effect. Infected cells and media are then harvested after viral replication is complete (generally, 7-10 days post-transfection).

[0059] It is to be noted that various alternative adenoviral serotypes can be developed in accordance with the disclosed methods and, particularly, alternative adenoviral serotype vectors that were previously unable to be propagated or very inefficiently propagated utilizing existing adenoviral production cell lines based on subgroup C complementing E1 sequence. The various adenoviral vectors that can be developed in accordance with the instant methods include adenoviral vectors of subgroups A-F (for instance, serotypes of subgroups A, B (e.g., serotypes 11, 14, 16, 21, 34 and 35), C (e.g., serotypes 2 and 5), D (e.g., serotypes 24, 26 and 36), E (e.g., serotype 4) and F.

[0060] In preferred embodiments, the various non-group C family members can be developed with heterologous E4 supplied from a subgroup C member such as adenovirus serotype 5. Particular embodiments of the instant invention utilize a development scheme wherein the heterologous E4 protein is derived from a wildtype adenovirus serotype 5 sequence; see, e.g., a viral sequence which has been deposited with the American Type Culture Collection (“ATCC”) under ATCC Deposit No. VR-5 (for which a transcription map can be found in FIG. 1). A particular example of this type of embodiment is wherein an adenovirus of subgroup B (or any non-C subgroup) comprising heterologous E4 proteins in cis from Ad5 is propagated in Ad5 E1-complementing cell lines, for instance, PER.C6™ or 293. Applicants have, in fact, successfully propagated E1-serotypes 10, 24, 34, and 35 via use of this particular embodiment.

[0061] One of skill in the art can readily identify alternative adenovirus serotypes (e.g., alternative serotypes of subgroups A, B (e.g., serotypes 11, 14, 16, 21, 34 and 35), C, (e.g., serotypes 2 and 5), D (e.g., serotypes 24, 26 and 36), E (e.g., serotype 4) and F) for the supply of the heterologous E4 protein. As long as the heterologous E4 region (or portion thereof comprising ORF6) of the vector is native to a virus of the same or highly similar serotype as the E1 region of the complementing cell line, the methods of the instant invention are widely applicable to the propagation and rescue of adenovirus of all serotypes. In light of the present disclosure, one can readily envision, for instance, how a complementing cell line based on a non-subgroup C adenovirus (e.g., the Ad35 cell line of EP 1 054 064 A1) can be utilized to propagate a virus of an adenoviral vector of subgroup C (e.g., adenovirus serotype 5) provided that the appropriate nucleic acid sequence encoding an E4 protein provided in cis is native to a virus of the same or highly similar serotype as that of the E1 expressed by the complementing cell line (i.e., an Ad35 E4 protein).

[0062] Complementing cell lines of use in the instant invention are available in the art and are not limited to any specific type. The critical feature, again, is that the heterologous segment of E4-encoding nucleic acid sequence provided in cis to the replication-defective vector being propagated be native to a virus of the same or highly similar serotype as the E1 expressed by the complementing cell line. One aspect of the instant invention employs E1-complementing cell lines wherein the expressed E1 is of serotype 5; e.g., PER.C6™ and 293 cell lines. Both these cell lines express the adenoviral E1 gene product. PER.C6™ is described in Fallaux et al., 1998 Human Gene Therapy 9:1909-1917, hereby incorporated by reference. 293 cell lines are described in Graham et al., 1977 J. Gen. Virol. 36:59-72, hereby incorporated by reference.

[0063] Another aspect of the instant invention are the adenoviral vectors of any serotype falling with adenoviral subgroups A, B, C, D, E and F (for instance, alternative serotypes of subgroups A, B (e.g., serotypes 11, 14, 16, 21, 34 and 35), C (e.g., serotype 2), D (e.g., serotypes 24, 26 and 36), E (e.g., serotype 4) and F) which are modified to contain a non-native E4-encoding nucleic acid sequence in cis which comprises, in whole or in part, nucleic acid sequence encoding open reading frame 6 (ORF6). Virus in accordance with this description can be propagated in accordance with the above-described methods and rescued using any suitable means known in the art.

[0064] Another aspect of the instant invention is a vector in accordance with the instant invention which comprises a heterologous passenger gene in addition to that of the heterologous E4 nucleic acid sequence. In specific embodiments, the passenger gene encodes an antigen.

[0065] As one of ordinary skill in the art will appreciate, the instant methods are not limited by the heterologous gene that can be incorporated. The instant invention relates generally to a means by which to propagate multiple serotypes of adenovirus in a single complementing cell line and the recombinant virus that make the process possible. In preferred embodiments, the passenger gene is incorporated into the E1 deletion. In alternatively preferred embodiments, the passenger gene is inserted in an E3-deleted region. The position of the passenger gene, as one of ordinary skill in the art will appreciate, can be varied according to the specific complementing cell utilized and the specific deletions present within the replication-defective adenovirus genome.

[0066] In specific embodiments the passenger gene can encode an HIV-1 antigen, and in more preferred embodiments selected from the group consisting of genes encoding HIV-1 gag, pol, nef and env. In alternative embodiments, the passenger gene can be a reporter gene, such as secreted alkaline phosphatase (SEAP).

[0067] The passenger gene preferably exists in the form of an expression cassette. A gene expression cassette preferably comprises (a) a nucleic acid sequence encoding a protein of interest; (b) a promoter operatively linked to the nucleic acid sequence encoding the protein; and (c) a transcription termination sequence. The transcriptional promoter of the adenoviral vector is preferably recognized by an eukaryotic RNA polymerase. In a preferred embodiment, the promoter is a “strong” or “efficient” promoter. An example of a strong promoter is the immediate early human cytomegalovirus promoter (Chapman et al., 1991 Nucl. Acids Res. 19:3979-3986), which is hereby incorporated by reference), in certain embodiments without intronic sequences. Those skilled in the art, however, will appreciate that any of a number of other known promoters, such as the strong immunoglobulin, or other eukaryotic gene promoters may also be used, including the EF1 alpha promoter, the murine CMV promoter, Rous sarcoma virus (RSV) promoter, SV40 early/late promoters and the beta-actin promoter.

[0068] The promoter may comprise a regulatable sequence such as the Tet operator sequence. This is extremely useful, for example, in cases where the gene products are affecting a result other than that desired and repression is sought.

[0069] Transcription termination sequences can also be utilized within the gene expression cassettes. Preferred termination sequences are, for instance, the bovine growth hormone terminator/polyadenylation signal (bGHpA) and the short synthetic polyA signal (SPA) of 50 nucleotides in length, defined as follows: AATAAAAGATCTTTATTTTCATTAGATCTGTGTGTTGGT-TTTTTGTGTG (SEQ ID NO:4).

[0070] Further embodiments incorporate a leader or signal peptide into the transgene. A preferred leader is that from the tissue-specific plasminogen activator protein, tPA.

[0071] The following non-limiting Examples are presented to better illustrate the invention.

EXAMPLE 1

[0072] Construction and Rescue

[0073] An E1-Ad35-based pre-adenovirus plasmid was constructed in order to determine whether an E1-Ad35 vector (a representative group B serotype) could be propagated in a group C E1-complementing cell line. The general strategy used to recover Ad35 as a bacterial plasmid is illustrated in FIG. 3. Cotransformation of BJ5183 bacteria with purified wild-type Ad35 viral DNA and a second DNA fragment termed the Ad35 ITR cassette resulted in the circularization of the viral genome by homologous recombination. The ITR cassette contains sequences from the right (bp 34419 to 34793) and left (bp 4 to 456 and bp 3403 to 3886) end of the Ad35 genome (see FIGS. 2A-1 to 2A-10) separated by plasmid sequences containing a bacterial origin of replication and an Ampicillin resistance gene. The ITR cassette contains a deletion of E1 sequences from Ad5 457 to 3402 with a unique Swa I site located in the deletion. The Ad35 sequences in the ITR cassette provide regions of homology with the purified Ad35 viral DNA in which recombination can occur. The ITR cassette was also designed to contain unique restriction enzyme sites (Pme I) located at the end of the viral ITR's so that digestion will release the Ad35 genome from plasmid sequences. Potential clones were screened by restriction analysis and one clone was selected as pAd35ΔE1. Pre-Adenovirus plasmid pAd35ΔE1 contains Ad35 sequences from 4 to 456 and bp 3403 to 34793.

[0074] To determine if pre-adenovirus plasmid pAd35ΔE1 could be rescued into virus and propagated in a group C E1 complementing cell line, the plasmid was digested with Pme I and transfected into a T-25 flask of PER.C6 cells using the calcium phosphate co-precipitation technique. Pme I digestion releases the viral genome from the plasmid sequences allowing viral replication to occur after entry into 293 cells. Viral cytopathic effect (CPE), indicating that virus replication and amplification is occurring, was never observed. Cells and media from the transfection were harvested at 14 days post transfection, freeze-thawed three times, clarified by centrifugation and used to infect new PER.C6 cells but no virus was ever amplified. Following multiple attempts, we have been unable to rescue and amplify pAd35ΔE1 in PER.C6 cells.

EXAMPLE 2

[0075] Insertion of Ad5 Orf 6 and Ad5 E4 into the Ad5 Genome

[0076] To refine the strategy of including Ad5 Orf6 in the genome of an alternative serotype so that propagation could take place in a Ad5/group C complementing cell line four additional strategies were developed. In the first strategy, the entire alternative serotype E4 region (not including the E4 promoter) was deleted and replaced with Ad5 Orf6. In the second strategy, just the alternative serotype Orf6 gene was deleted and replaced with Ad5 Orf6. In the third strategy, the entire alternative serotype E4 coding region (not including the E4 promoter) was deleted and replaced with the Ad5 E4 coding region (not including the Ad5 E4 promoter) and, in the final strategy, the entire alternative serotype E4 coding and promoter region was deleted and replaced with the Ad5 E4 promoter and coding region. The configuration of the E4 regions generated by the four strategies is diagramed in FIG. 4. For each of these strategies the desired pre-Adenovirus plasmid was generated by bacterial recombination. Cotransformation of BJ 5183 bacteria with purified wild-type viral DNA and the appropriately constructed ITR cassette resulted in the circularization of the viral genome by homologous recombination. The construction of each pre-Ad plasmid, based on Ad35, is outlined below:

[0077] To construct pAd35ΔE1ΔE4Ad5Orf6 (An Ad35 pre-Ad plasmid containing an E1 deletion and an E4 deletion substituted with Ad5 Orf6), an Ad35 ITR cassette was constructed containing sequences from the right (bp 31599 to 31913 and bp 34419 to 34793) and left (bp 4 to 456 and bp 3403 to 3886) end of the Ad35 genome separated by plasmid sequences containing a bacterial origin of replication and an ampicillin resistance gene. These four segments were generated by PCR and cloned sequentially into pNEB193, generating pNEBAd35-4. Next the Ad5 Orf6 open reading frame was generated by PCR and cloned between Ad35 bp 31913 and 34419 generating pNEBAd35-4Ad5Orf6 (the ITR cassette). PNEB193 is a commonly used commercially available cloning plasmid (New England Biolabs cat# N3051 S) containing a bacterial origin of replication, ampicillin resistance gene and a multiple cloning site into which the PCR products were introduced. The ITR cassette contains a deletion of E1 sequences from Ad35 bp 457 to 3402 with a unique Swa I restriction site located in the deletion and an E4 deletion from Ad35 bp 31912 to 34418 into which Ad5 Orf6 was introduced in an E4 parallel orientation. In this construct, Ad5Orf6 expression is driven by the Ad35 E4 promoter. The Ad35 sequences (bp 31599 to 31913 and bp 3403 to 3886) in the ITR cassette provide regions of homology with the purified Ad35 viral DNA in which bacterial recombination can occur following cotransformation into BJ 5183 bacteria (FIG. 5). The ITR cassette was also designed to contain unique restriction enzyme sites (PmeI) located at the end of the viral ITR's so that digestion will release the recombinant Ad35 genome from plasmid sequences. Potential clones were screened by restriction analysis and one clone was selected as pAd35ΔE1ΔE4Ad5Orf6. Pre-Adenovirus plasmid pAd35ΔE1ΔE4Ad5Orf6 contains Ad35 sequences from bp 4 to 456; bp 3403 to bp 31913 and bp 34419 to bp 34793 with Ad5Orf6 cloned between bp 31913 and bp 34419.

[0078] To construct pAd35ΔE1ΔOrf6Ad5Orf6 (An Ad35 pre-Ad plasmid containing an E1 deletion and a deletion of E4 Orf6 substituted with Ad5 Orf6), an Ad35 ITR cassette was constructed containing sequences from the right (bp 31599 to 32081 and bp 32990 to 34793) and left (bp 4 to 456 and bp 3403 to 3886) end of the Ad35 genome separated by plasmid sequences containing a bacterial origin of replication and an ampicillin resistance gene. These four segments were generated by PCR and cloned sequentially into pNEB193, generating pNEBAd35-10. Next the Ad5 Orf6 open reading frame was generated by PCR and cloned between Ad35 bp 32081 and 32990 generating pNEBAd35-10Ad5Orf6 (the ITR cassette). PNEB193 is a commonly used commercially available cloning plasmid (New England Biolabs cat# N3051S) containing a bacterial origin of replication, ampicillin resistance gene and a multiple cloning site into which the PCR products were introduced. The ITR cassette contains a deletion of E1 sequences from Ad35 bp 457 to 3402 with a unique Swa I restriction site located in the deletion and a deletion of E4 Orf6 from Ad35 bp 32082 to 32989 into which Ad5 Orf6 was introduced in an E4 parallel orientation. In this construct, Ad5Orf6 expression is driven by the Ad35 E4 promoter. The Ad35 sequences (bp 31599 to 32081 and bp 3403 to 3886) in the ITR cassette provide regions of homology with the purified Ad35 viral DNA in which bacterial recombination can occur following cotransformation into BJ 5183 bacteria. The ITR cassette was also designed to contain unique restriction enzyme sites (Pme I) located at the end of the viral ITR's so that digestion will release the recombinant Ad35 genome from plasmid sequences. Potential clones were screened by restriction analysis and one clone was selected as pAd35ΔE1ΔOrf6Ad5Orf6. Pre-Adenovirus plasmid pAd35ΔE1ΔOrf6Ad5Orf6 contains Ad35 sequences from bp 4 to 456; bp 3403 to bp 32081 and bp 32990 to bp 34793 with Ad5Orf6 cloned between bp 32081 and bp 32990.

[0079] To construct pAd35ΔE1ΔE4Ad5E4 (An Ad35 pre-Ad plasmid containing an E1 deletion and a deletion of E4 substituted with Ad5 E4), an Ad35 ITR cassette was constructed containing sequences from the right (bp 31599 to 31838 and bp 34419 to 34793) and left (bp 4 to 456 and bp 3403 to 3886) end of the Ad35 genome separated by plasmid sequences containing a bacterial origin of replication and an ampicillin resistance gene. These four segments were generated by PCR and cloned sequentially into pNEB193, generating pNEBAd35-7. Next the Ad5 E4 coding region was generated by PCR and cloned between Ad35 bp 31838 and 34419 generating pNEBAd35-7Ad5E4-2 (the ITR cassette). PNEB193 is a commonly used commercially available cloning plasmid (New England Biolabs cat# N3051 S) containing a bacterial origin of replication, ampicillin resistance gene and a multiple cloning site into which the PCR products were introduced. The ITR cassette contains a deletion of E1 sequences from Ad35 bp 457 to 3402 with a unique Swa I restriction site located in the deletion and an E4 deletion from Ad35 bp 31839 to 34418 into which the Ad5 E4 coding region was introduced in an E4 parallel orientation. In this construct, the Ad5 E4 region is expressed using the Ad35 E4 promoter. The Ad35 sequences (bp 31599 to 31838 and bp 3403 to 3886) in the ITR cassette provide regions of homology with the purified Ad35 viral DNA in which bacterial recombination can occur following cotransformation into BJ 5183 bacteria. The ITR cassette was also designed to contain unique restriction enzyme sites (Pme I) located at the end of the viral ITR's so that digestion will release the recombinant Ad35 genome from plasmid sequences. Potential clones were screened by restriction analysis and one clone was selected as pAd35ΔE1ΔE4Ad5E4. Pre-Adenovirus plasmid pAd35ΔE1ΔE4Ad5E4 contains Ad35 sequences from bp 4 to 456; bp 3403 to bp 31838 and bp 34419 to bp 34793 with the Ad5 E4 coding region (Ad 5 bp 32914 to bp 35523) cloned between bp 31838 and bp 34419.

[0080] To construct pAd35ΔE1ΔE4Ad5PE4 (An Ad35 pre-Ad plasmid containing an E1 deletion and a deletion of E4 coding region and promoter substituted with Ad5 E4 coding region and promoter), an Ad35 ITR cassette was constructed containing sequences from the right (bp 31599 to 31838 and bp 34660 to 34793) and left (bp 4 to 456 and bp 3403 to 3886) end of the Ad35 genome separated by plasmid sequences containing a bacterial origin of replication and an ampicillin resistance gene. These four segments were generated by PCR and cloned sequentially into pNEB193, generating pNEBAd35-8. Next the Ad5 E4 promoter and coding region was generated by PCR and cloned between Ad35 bp 31838 and 34660 generating pNEBAd35-8Ad5E4PC (the ITR cassette). PNEB193 is a commonly used commercially available cloning plasmid (New England Biolabs cat# N3051S) containing a bacterial origin of replication, ampicillin resistance gene, and a multiple cloning site into which the PCR products were introduced. The ITR cassette contains a deletion of E1 sequences from Ad35 bp 457 to 3402 with a unique Swa I restriction site located in the deletion and an E4 deletion from Ad35 bp 31839 to 34659 into which the Ad5 E4 promoter and coding region was introduced in an E4 parallel orientation. In this construct, the Ad5 E4 region is expressed using the Ad5 E4 promoter. The Ad35 sequences (bp 31599 to 31838 and bp 3403 to 3886) in the ITR cassette provide regions of homology with the purified Ad35 viral DNA in which bacterial recombination can occur following cotransformation into BJ 5183 bacteria. The ITR cassette was also designed to contain unique restriction enzyme sites (Pme I) located at the end of the viral ITR's so that digestion will release the recombinant Ad35 genome from plasmid sequences. Potential clones were screened by restriction analysis and one clone was selected as pAd35ΔE1ΔE4Ad5PE4. Pre-Adenovirus plasmid pAd35ΔE1ΔE4Ad5PE4 contains Ad35 sequences from bp 4 to 456; bp 3403 to bp 31838 and bp 34660 to bp 34793 with the Ad5 E4 promoter and coding region (Ad 5 bp 32914 to bp 35826) cloned between bp 31838 and bp 34660.

EXAMPLE 3

[0081] Rescue of pAd35ΔE1ΔE4Ad5Orf6, pAd35ΔE1ΔOrf6Ad5Orf6, pAd35ΔE1ΔE4Ad5E4 and pAd35ΔE1ΔE4Ad5PE4 into Virus

[0082] In order to determine if pre-adenovirus plasmids pAd35ΔE1ΔE4Ad5Orf6, pAd35ΔE1ΔOrf6Ad5Orf6, pAd35ΔE1ΔE4Ad5E4 and pAd35ΔE1ΔE4Ad5PE4 could be rescued into virus and propagated in a group C E1 complementing cell line, the plasmids were each digested with Pme I and transfected into T-25 flasks of PER.C6 cells using the calcium phosphate co-precipitation technique; Cell Phect Transfection Kit, Amersham Pharmacia Biotech Inc. PmeI digestion releases the viral genome from plasmid sequences allowing viral replication to occur after cell entry. Viral cytopathic effect (CPE), indicating that virus replication and amplification was occurring, was observed for all construct. When CPE was complete, approximately 7-10 days post transfection, the infected cells and media were harvested, freeze/thawed three times and the cell debris pelleted by centrifugation. Approximately 1 ml of the cell lysate was used to infect aT-225 flasks of PER.C6 cells at 80-90% confluence. Once CPE was reached, infected cells and media were harvested, freeze/thawed three times and the cell debris pelleted by centrifugation. Clarified cell lysates were then used to infect 2-layer NUNC cell factories of PER.C6 cells. Following complete CPE the virus was purified by ultracentrifugation on CsCl density gradients. In order to verify the genetic structure of the rescued viruses, viral DNA was extracted using pronase treatment followed by phenol chloroform extraction and ethanol precipitation. Viral DNA was then digested with HindIII and treated with Klenow fragment to end-label the restriction fragments with P33-dATP. The end-labeled restriction fragments were then size-fractionated by gel electrophoresis and visualized by autoradiography. The digestion products were compared with the digestion products of the corresponding pre-Adenovirus plasmid (that had been digested with Pme1/HindIII prior to labeling) from which they were derived. The expected sizes were observed, indicating that the viruses had been successfully rescued.

EXAMPLE 4

[0083] Insertion of an Expression Cassette into pAd35ΔE1ΔE4Ad5Orf6, pAd35ΔE1ΔOrf6Ad5Orf6, pAd35ΔE1ΔE4Ad5E4 and pAd35ΔE1ΔE4Ad5PE4

[0084] In order to introduce a gag or SEAP expression cassette into the E1 region of the various Ad35 pre-Adenovirus plasmids described above (pAd35ΔE1ΔE4Ad5Orf6, pAd35ΔE1ΔOrf6Ad5Orf6, pAd35ΔE1ΔE4Ad5E4 and pAd35ΔE1ΔE4Ad5PE4) bacterial recombination was again used. A gag expression cassette consisting of the following: 1) the immediate early gene promoter from the human cytomegalovirus, 2) the coding sequence of the human immunodeficiency virus type 1 (HIV-1) gag (strain CAM-1; 1526 bp) gene, and 3) the bovine growth hormone polyadenylation signal sequence (FIG. 6), was cloned into the E1 deletion in Ad35 shuttle plasmid, pNEBAd35-2 (a precursor to the Ad35 ITR cassettes described above), generating pNEBAd35CMVgagBGHpA. pNEBAd35-2 contains Ad35 sequences from the left end of the genome (bp 4 to 456 and bp 3403 to 3886) with a unique SwaI site between bp 456 and 3403 at the position of the deletion. The gag expression cassette was obtained from a previously constructed shuttle plasmid by EcoRI digestion. Following the digestion the desired fragment was gel purified, treated with Klenow to obtain blunt ends and cloned into the SwaI site in pNEBAd35-2. This cloning step resulted in the gag expression cassette being cloned into the E1 deletion between bp 456 and 3403 in the E1 parallel orientation. The shuttle vector containing the gag transgene was digested to generate a DNA fragment consisting of the gag expression cassette flanked by Ad35 bp 4 to 456 and bp 3403 to 3886 and the fragment was purified after electrophoresis on an agarose gel. Cotransformation of BJ 5183 bacteria with the shuttle vector fragment and one of the Ad35 pre-Ad plasmids (pAd35ΔE1ΔE4Ad5Orf6, pAd35ΔE1ΔOrf6Ad5Orf6, pAd35ΔE1ΔE4Ad5E4, pAd35ΔE1ΔE4Ad5PE4), linearized in the E1 region by digestion with Swa I, resulted in the generation of corresponding Ad35 gag-containing pre-Adenovirus plasmids (pAd35ΔE1gagΔE4Ad5Orf6, pAd35ΔE1 gagΔOrf6Ad5Orf6, pAd35ΔE1gagΔE4Ad5E4, and pAd35ΔE1gagΔE4Ad5PE4) by homologous recombination. Potential clones were screened by restriction analysis.

[0085] A similar strategy was used to generate Ad35 pre-Ad plasmids containing a SEAP expression cassette. In this case a SEAP expression cassette consisting of: 1) the immediate early gene promoter from the human cytomegalovirus, 2) the coding sequence of the human placental SEAP gene, and 3) the bovine growth hormone polyadenylation signal sequence (FIG. 7) was cloned into the E1 deletion in Ad35 shuttle plasmid, pNEBAd35-2, generating pNEBAd35CMVSEAPBGHpA. The SEAP expression cassette was obtained from a previously constructed shuttle plasmid by EcoRI digestion. Following the digestion the desired fragment was gel purified, treated with Klenow to obtain blunt ends and cloned into the SwaI site in pNEBAd35-2. The transgene was then recombined into the various Ad35 backbones generating pAd35ΔE1SEAPΔE4Ad5Orf6, pAd35ΔE1SEAPΔOrf6Ad5Orf6, pAd35ΔE1SEAPΔE4Ad5E4, and pAd35ΔE1SEAPΔE4Ad5PE4 as described above for the gag transgene. All pre-Ad plasmids were rescued into virus and expanded to prepare CsCl purified stocks as described above.

EXAMPLE 5

[0086] In vivo Transgene Expression

[0087] A. Immunization

[0088] Female mice were between 4-10 weeks old. The total dose of each vaccine was suspended in 0.1 mL of buffer. The vectors were given to both quadriceps of each animals with a volume of 50 μL per quad and using 0.3-mL 28G1/2 insulin syringes (Becton-Dickinson, Franklin Lakes, N.J.). The rhesus macaques and African green monkeys were between 2-5 kg in weight. For the primates, the total dose of each vaccine was suspended in 1 mL of buffer. The monkeys were anesthetized (ketamine/xylazine mixture) and the vaccines were delivered i.m. in 0.5-mL aliquots into two muscle sites using tuberculin syringes (Becton-Dickinson, Franklin Lakes, N.J.). Serum samples were collected at defined intervals and stored frozen until the assay date. All animal care and treatment were in accordance with standards approved by the Institutional Animal Care and Use Committee according to the principles set forth in the Guide for Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council.

[0089] B. SEAP Assay

[0090] Serum samples were analyzed for circulating SEAP levels using TROPIX phospha-light chemiluminescent kit (Applied Biosystems Inc). Duplicate 5 μL aliquots of each serum were mixed with 45 μL of kit-supplied dilution buffer in a 96-well white DYNEX plate. Serially diluted solutions of a human placental alkaline phosphatase (Catalog no. M5905, Sigma, St. Louis, Mo.) in 10% naïve monkey or mouse serum served to provide the standard curve. Endogenous SEAP activity in the samples was inactivated by heating the well for 30 minutes at 65° C. Enzymatic SEAP activities in the samples were determined following the procedures described in the kit. Chemiluminescence readings (in relative light units) were recorder using DYNEX luminometer. RLU readings are converted to ng/mL SEAP using a log-log regression analyses.

[0091] C. Rodent Results

[0092] In the first mouse experiment, cohorts of 5 C3H/HeN mice were given single intramuscular injections of one of the following vectors: (1) 10{circumflex over ( )}10 vp Ad35ΔE1SEAPΔE4Ad5Orf6; (2) 10{circumflex over ( )}10 vp Ad35ΔE1SEAPΔE3ΔE4Ad5Orf6; or (3) 10{circumflex over ( )}10 vp Ad35ΔE1SEAP. Serum samples prior to and after the injection were analyzed for circulating SEAP activities and the results are shown in FIG. 8. Results indicate that (1) the Ad35 constructs are all capable of expressing the SEAP transgene and that (2) the introduction of Ad5Orf6 sequence where the deleted Ad35E4 was did not significantly affect the transgene expression relative to Ad35ΔE1SEAP. Ad35ΔE1SEAPΔE3ΔE4Ad5Orf6 also yielded a similar expression profile as Ad35ΔE1SEAP. The levels of SEAP in the serum dropped after day 2 and were at background levels by day 12.

[0093] The second mouse experiment evaluates the effect of a full Ad5E4 replacement instead of an Ad5Orf6 substitution for the Ad35 E4 cassette. Here, cohorts of 5 C3H/HeN mice were given single intramuscular injections of one of the following vectors: (1) 10{circumflex over ( )}10 vp MRKAd5-SEAP; (2) 10{circumflex over ( )}9 vp MRKAd5-SEAP; (3) 10{circumflex over ( )}10 vp Ad35ΔE1SEAPΔE4Ad5Orf6; (4) 10{circumflex over ( )}10 vp Ad35ΔE1SEAPΔE4Ad5E4; or (5) 10{circumflex over ( )}10 vp Ad35ΔE1SEAPΔE4Ad5PE4. The introduction of Ad5E4 or Ad5PE4 resulted in comparable if not, slightly improved expression levels compared to the vector with the Ad5Orf6 insertion (FIG. 9). The peak levels for the Ad35 constructs are lower than those produced by Ad5SEAP (at least 10-fold).

[0094] D. Primate Results

[0095] Cohorts of 3 rhesus macaques were given single intramuscular injections of one of the following vectors: (1) 10{circumflex over ( )}11 vp MRKAd5-SEAP; (2) 10{circumflex over ( )}9 vp MRKAd5-SEAP; or (3) 10{circumflex over ( )}11 vp Ad35ΔE1SEAPΔE4Ad5Orf6. Serum samples prior to and after the injection were analyzed for circulating SEAP activities and the results for the individual monkeys are shown in FIGS. 10A-B. Results indicate that the peak level of SEAP product produced by the alternative adenovirus serotype was lower than but were within 3-fold of that of MRKAd5SEAP at the same high dose level of 10{circumflex over ( )}11 vp. The levels observed from the Ad35 vector were about 50-fold higher than those observed using 10{circumflex over ( )}9 vp of MRKAd5SEAP. The levels of SEAP in the serum dropped after day 10 and were close to background as early as day 15.

[0096] A separate experiment using African green monkeys was conducted to examine the effect of the additional E3 deletion or the full Ad5E4 substitution on in vivo gene expression. In here, cohorts of 2-3 African green macaques were given single intramuscular injections of one of the following vectors: (1) 10{circumflex over ( )}11 vp MRKAd5-SEAP; (2) 10{circumflex over ( )}10 vp MRKAd5-SEAP; (3) 10{circumflex over ( )}9 vp MRKAd5-SEAP; (4) 10{circumflex over ( )}10 vp Ad35ΔE1SEAPΔE4Ad5Orf6; (5) 10{circumflex over ( )}10 vp Ad35ΔE1SEAPΔE3ΔE4Ad5Orf6; or (6) 10{circumflex over ( )}10 vp Ad35ΔE1SEAPΔE4Ad5E4. Results (FIG. 11) indicate that the peak levels of SEAP product produced by Ad35ΔE1SEAPΔE3ΔE4Ad5Orf6 and Ad35ΔE1SEAPΔE4Ad5E4 were comparable if not, slightly improved compared to Ad35ΔE1SEAPΔE4Ad5Orf6.

EXAMPLE 6

[0097] In Vivo Immunogenicity

[0098] A. Immunization

[0099] Cohorts of 3-6 animals were given intramuscular injections at wk 0 and wk 4 of either of the following constructs: (1) 10{circumflex over ( )}11 vp MRKAd5-HIV1 gag; or (2) 10{circumflex over ( )}11 vp of Ad35ΔE1gagΔE4Ad5Orf6. Rhesus macaques were between 3-10 kg in weight. In all cases, the total dose of each vaccine was suspended in 1 mL of buffer. The macaques were anesthetized (ketamine/xylazine) and the vaccines were delivered i.m. in 0.5-mL aliquots into both deltoid muscles using tuberculin syringes (Becton-Dickinson). Sera and peripheral blood mononuclear cells (PBMC) were prepared from blood samples collected at several time points during the immunization regimen. All animal care and treatment were in accordance with standards approved by the Institutional Animal Care and Use Committee according to the principles set forth in the Guide for Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council.

[0100] B. ELISPOT Assay

[0101] The IFN-γ ELISPOT assays for rhesus macaques were conducted following a previously described protocol (Allen et al., 2001 J. Virol. 75(2):738-749), with some modifications. For antigen-specific stimulation, a peptide pool was prepared from 20-aa peptides that encompass the entire HIV-1 gag sequence with 10-aa overlaps (Synpep Corp., Dublin, Calif.). To each well, 50 μL of 2-4×10⁵ peripheral blood mononuclear cells (PBMCs) were added; the cells were counted using Beckman Coulter Z2 particle analyzer with a lower size cut-off set at 80 femtoliters (“fL”). Either 50 μL of media or the gag peptide pool at 8 μg/mL concentration per peptide was added to the PBMC. The samples were incubated at 37° C., 5% CO₂ for 20-24 hrs. Spots were developed accordingly and the plates were processed using custom-built imager and automatic counting subroutine based on the ImagePro platform (Silver Spring, Md.); the counts were normalized to 10⁶ cell input.

[0102] C. Intracellular Cytokine Staining

[0103] To 1 ml of 2×10⁶ PBMC/mL in complete RPMI media (in 17×100 mm round bottom polypropylene tubes (Sarstedt, Newton, N.C.)), anti-hCD28 (clone L293, Becton-Dickinson) and anti-hCD49d (clone L25, Becton-Dickinson) monoclonal antibodies were added to a final concentration of 1 μg/mL. For gag-specific stimulation, 10 μL of the peptide pool (at 0.4 mg/mL per peptide) were added. The tubes were incubated at 37° C. for 1 hr., after which 20 μL of 5 mg/mL of brefeldin A (Sigma) were added. The cells were incubated for 16 hr at 37° C., 5% CO₂, 90% humidity. 4 mL cold PBS/2% FBS were added to each tube and the cells were pelleted for 10 min at 1200 rpm. The cells were re-suspended in PBS/2% FBS and stained (30 min, 4° C.) for surface markers using several fluorescent-tagged mAbs: 20 μL per tube anti-hCD3-APC, clone FN-18 (Biosource); 20 μL anti-hCD8-PerCP, clone SK1 (Becton Dickinson, Franklin Lakes, N.J.); and 20 μL anti-hCD4-PE, clone SK3 (Becton Dickinson). Sample handling from this stage was conducted in the dark. The cells were washed and incubated in 750 μL 1×FACS Perm buffer (Becton Dickinson) for 10 min at room temperature. The cells were pelleted and re-suspended in PBS/2% FBS and 0.1 μg of FITC-anti-hIFN-γ, clone MD-1 (Biosource) was added. After 30 min incubation, the cells were washed and re-suspended in PBS. Samples were analyzed using all four color channels of the Becton Dickinson FACSCalibur instrument. To analyze the data, the low side- and forward-scatter lymphocyte population was initially gated; a common fluorescence cut-off for cytokine-positive events was used for both CD4⁺ and CD8⁺ populations, and for both mock and gag-peptide reaction tubes of a sample.

[0104] D. Results

[0105] PBMCs collected at regular 4-wk intervals were analyzed in an ELISPOT assay. Results (Table 1) indicate that the Ad35ΔE1gagΔE4Ad5Orf6 is able to induce in non-human primates significant levels of gag-specific T cells. After a single dose (wk 4), the Ad35-induced responses were about 5-fold lower than that of MRKAd5-HIV1 gag. After the second dose (wk 8), the responses between both cohorts were comparable; the differences became pronounced in the succeeding time points. TABLE 1 Gag-specific T cell response in monkeys immunized with MRKAd5-HIV1gag and Ad35ΔE1gagΔE4Ad5Orf6. Shown is the number of spot-forming cells per million PBMC following incubation in the absence (mock) or presence of Gag H peptide pool. The H pool consisted of 20-aa peptide overlapping by 10 aa and encompassing the entire gag sequence. Vaccine Monkey Pre Wk 4 Wk 8 Wk 12 Wk 16 Grp Wk 0, Wk 4 ID Mock Gag H Mock Gag H Mock Gag H Mock Gag H Mock Gag H 1 MRKAd5-HIV1 gag 00C018 1 5 13 1025 0 824 3 753 1 533 10{circumflex over ( )}11 vp 00C034 0 4 5 219 5 404 0 491 1 350 00C058 4 4 3 1086 0 440 0 439 0 599 2 Ad35ΔE1gagΔE4Ad5Orf6 00D045 1 1 3 168 5 645 4 178 0 91 10{circumflex over ( )}11 vp 00D067 1 4 5 89 0 103 0 76 0 19 00D068 1 4 10 34 5 365 3 143 0 95 00D054 3 15 10 195 0 501 3 350 0 124 00D075 3 5 18 275 13 716 3 158 0 103 00D073 14 26 1 241 3 485 3 278 0 148 3 Naïve 00D087 1 1 3 3 8 54 3 5 3 1

[0106] Intracellular IFN-γ staining analyses of PBMC collected at wk 8 suggest that the Ad35-based vaccine is able to induce both HIV-specific CD4+ and CD8+ T cells (Table 2). TABLE 2 Characterization of the gag-specific T cells in monkeys immunized with MRKAd5-HIV1gag and Ad35ΔE1gagΔE4Ad5Orf6. Shown are the percentages of CD3+ T cells that are either gag-specific CD4+ or gag-specific CD8+ cells. These values were corrected for mock values (<0.02%). Wk 8 Vaccine Monkey % CD4 + % CD8 + Grp Wk 0, Wk 4 ID CD3+ CD3+ 1 MRKAd5-HIV1 gag 00C018 0.08 0.37 10{circumflex over ( )}11 vp 00C034 0.09 0.06 00C058 0.03 0.21 2 Ad35ΔE1gagΔE4Ad5Orf6 00D045 0.06 0.08 10{circumflex over ( )}11 vp 00D067 0.02 0.02 00D068 0.15 0.02 00D054 0.05 0.08 00D075 0.08 0.05 00D073 0.09 0.06

[0107] In a separate experiment, 3 different Ad35 constructs expressing HIV-1 gag were evaluated for their immunogenicity in macaques. Here, cohorts of 3 macaques were given immunizations at wk 0 and 4 of either of the following vectors: (1) 10{circumflex over ( )}10 vp Ad35ΔE1gagΔE4Ad5Orf6; (2) 10{circumflex over ( )}10 vp Ad35ΔE1gagΔE3ΔE4Ad5Orf6; or (3) 10{circumflex over ( )}10 vp Ad35ΔE1gagΔE4Ad5E4. The levels of T cell immunity induced by all 3 vectors were comparable at this stage (Table 2), suggesting that the additional E3 deletion or full Ad5E4 substitution does not appear to impair the immunogenic properties of the vector. TABLE 3 Gag-specific T cell response in monkeys immunized with several Ad35ΔE1ΔE4-based vectors. Shown is the number of spot-forming cells per million PBMC following incubation in the absence (mocK0 or presence of Gag H peptide pool. The H pool consisted of 20-aa peptide overlapping by 10 aa and encompassing the entire gag sequence. Vaccine Monkey Pre Wk 4 Wk 8 Grp Wk 0, Wk 4 ID Mock Gag H Mock Gag H Mock Gag H 1 Ad35ΔE1gagΔE4Ad5Orf6 00C047 4 1 0 20 0 189 10{circumflex over ( )}10vp 00C157 8 5 1 81 1 833 00C078 3 1 0 46 4 349 2 Ad35ΔE1gagΔE3ΔE4Ad5Orf6 00C091 1 1 1 118 3 315 10{circumflex over ( )}10vp 00C122 3 0 0 31 1 138 00D177 3 3 1 45 1 64 3 Ad35ΔE1gagΔE4Ad5E4 00D018 3 19 29 120 23 193 10{circumflex over ( )}10vp 00D046 8 5 1 21 10 143 00D063 3 4 0 63 4 371 Naïve none 00D363 0 5 ND ND 0 0

EXAMPLE 7

[0108] Construction and Rescue of pAd24ΔE1.

[0109] An E1-Ad24-based pre-adenovirus plasmid was constructed in order to determine whether an E1-Ad24 vector (a representative group D serotype) could be propagated in an Ad5/group C E1-complementing cell line. Since at the time the vector construction was initiated the complete sequence of Ad24 (see FIGS. 16A-1 through 16A-10; subject of copending application serial No. 60/455,312, filed Mar. 17, 2003) was unknown we took advantage of some sequence homology between Ad24 and Ad17. The general strategy used to recover Ad24 as a bacterial plasmid is illustrated in FIG. 12 and described below. Cotransformation of BJ5183 bacteria with purified wild-type Ad24 viral DNA and a second DNA fragment termed the Ad 17 ITR cassette resulted in the circularization of the viral genome by homologous recombination. The ITR cassette contains sequences from the right (bp 34469 to 35098) and left (bp 4 to 414 and bp 3373 to 4580) end of the Ad17 genome (Accession No. AF108105) separated by plasmid sequences containing a bacterial origin of replication and an Ampicillin resistance gene. The ITR cassette contains a deletion of E1 sequences from Ad17 (bp 415 to 3372) with a unique Swa I site located in the deletion. The Ad17 sequences in the ITR cassette provide regions of homology with the purified Ad24 viral DNA in which recombination can occur. The ITR cassette was also designed to contain unique restriction enzyme sites (Pme I) located at the end of the viral ITR's so that digestion will release the Ad24 genome from plasmid sequences. Potential clones were screened by restriction analysis and one clone was selected as pAd24ΔE1. pAd24ΔE1 contains Ad17 sequences from bp 4 to 414 and from bp 3373 to 4580, Ad24 bp 4588 to 34529, and Ad17 bp 34469 to 35098 (bp numbers refer to the wt sequence for both Ad17 and Ad24). PAd24ΔE1 contains the coding sequences for all Ad24 virion structural proteins that constitute its serotype specificity. This approach can be used to circularize any group D serotype into plasmid form which has sufficient homology to Ad17.

[0110] To determine if pre-adenovirus plasmid pAd24ΔE1 could be rescued into virus and propagated in a group C E1 complementing cell line, the plasmid was digested with Pme I and transfected into a 6 cm dish of 293 cells using the calcium phosphate co-precipitation technique. Pme I digestion releases the viral genome from the plasmid sequences allowing viral replication to occur after entry into 293 cells. Viral cytopathic effect (CPE), indicating that virus replication and amplification is occurring, was very slow to arise. Following multiple attempts, we were successful at rescuing and amplifying Ad24ΔE1 but the virus grew to lower titers and took more passages to amplify than a similar Ad5 based vector. In order to verify the genetic structure of the virus, viral DNA was extracted using pronase treatment followed by phenol chloroform extraction and ethanol precipitation. Viral DNA was then digested with HindIII and treated with Klenow fragment to end-label the restriction fragments with P33-dATP. The end-labeled restriction fragments were then size-fractionated by gel electrophoresis and visualized by autoradiography. The digestion products were compared with the digestion products from the pre-plasmid (that had been digested with Pme1/HindIII prior to labeling). The expected sizes were observed, indicating that the virus had been successfully rescued.

EXAMPLE 8

[0111] Insertion of Ad5 Orf 6 into the E1 region of Ad24

[0112] In order to determine if the insertion of Ad5 E4 Orf6 into the Ad24 genome would allow more efficient propagation in a group C E1 complementing cell line we constructed an Ad24 based pre-adenovirus plasmid containing Ad5 Orf6 in the E1 region. In order to introduce Ad5 Orf6 in to the E1 region of pAd24ΔE1, bacterial recombination was used. An Ad5 Orf6 transgene consisting of the Ad5 Orf6 coding region flanked by the HCMV promoter and pA was cloned into the E1 deletion in an Ad 17 shuttle vector (a precursor to the Ad 17 ITR cassette). The Ad5 Orf6 transgene was cloned between bp 414 and 3373 in the E1 anti-parallel orientation. The shuttle vector containing the Ad5 Orf6 transgene was digested to generate a DNA fragment consisting of the transgene flanked by Ad17 sequences (bp 4 to 414 and bp 3373 to 4580) and the fragment was purified after electrophoresis on an agarose gel. Cotransformation of BJ 5183 bacteria with the shuttle vector fragment and pAd24ΔE1, which had been linearized in the E1 region by digestion with SwaI, resulted in the generation of pAd24ΔE1Ad5Orf6 by homologous recombination (FIG. 13). Potential clones were screened by restriction analysis and one clone was selected as pre-adenovirus plasmid pAd24ΔE1Ad5Orf6.

[0113] In order to determine if pre-adenovirus plasmid pAd24ΔE1Ad5Orf6 could be rescued into virus and propagated in an Ad5/group C E1 complementing cell line, pAd24ΔE1Ad5Orf6 was digested with Pme I and transfected into a 6 cm dish of 293 cells using the calcium phosphate co-precipitation technique. PmeI digestion releases the viral genome from plasmid sequences allowing viral replication to occur after entry into 293 cells. Once complete viral cytopathic effect (CPE) was observed at approximately 7-10 days post transfection, the infected cells and media were freeze/thawed three times and the cell debris pelleted. The virus was amplified in two additional passages in 293 cells and then purified from the final infection by ultracentrifugation on CsCl density gradients. In order to verify the genetic structure of the virus, viral DNA was extracted using pronase treatment followed by phenol chloroform extraction and ethanol precipitation. Viral DNA was then digested with HindIII and treated with Klenow fragment to end-label the restriction fragments with P33-dATP. The end-labeled restriction fragments were then size-fractionated by gel electrophoresis and visualized by autoradiography. The digestion products were compared with the digestion products from the pre-plasmid (that had been digested with Pme1/HindIII prior to labeling). The expected sizes were observed, indicating that the virus had been successfully rescued.

EXAMPLE 9

[0114] Insertion of Ad5 Orf6 into the E4 region of Ad24

[0115] To refine the strategy of including Ad5 Orf6 in the genome of an alternative serotype so that propagation could take place in an Ad5/group C complementing cell line two additional strategies were developed. In the first strategy, the entire alternative serotype E4 region (not including the E4 promoter) was deleted and replaced with Ad5 Orf6. In the second strategy, just the alternative serotype Orf6 gene was deleted and replaced with Ad5 Orf6. The configuration of the E4 regions generated by the two strategies is diagramed in FIG. 14. For each of these strategies the desired pre-Adenovirus plasmid was generated by bacterial recombination. Cotransformation of BJ 5183 bacteria with pAd24ΔOrf6BstZ17I and the appropriately constructed Ad24 E4 shuttle plasmid resulted in the generation of the desired Ad24 based pre-Ad plasmid. PAd24ΔOrf6BstZ17I, a derivative of pAd24ΔE1, was constructed so that the E4 region in the Ad24 pre-Ad plasmid could be easily modified using bacterial recombination. PAd24ΔOrf6BstZ17I contains a deletion in the E4 region from Ad24 bp 32373 to bp 33328 with a unique BstZ17I site located at the position of the deletion. The complete sequence of pAd24ΔOrf6BstZ17I consists of Ad17 sequences from bp 4 to 414 and from bp 3373 to 4580, Ad24 bp 4588 to 32372 and from 33329 to 34529, and Ad17 bp 34469 to 35098 (bp numbers refer to the wt sequence for both Ad17 and Ad24).

[0116] To construct pAd24ΔE1ΔE4Ad5Orf6 (An Ad24 pre-Ad plasmid containing an E1 deletion and a deletion of E4 substituted with Ad5 Orf6), an Ad24 E4 shuttle plasmid was constructed by digesting pAd24ΔE1 with PmeI and BsrGI and cloning the restriction fragment representing the E4 region (bp 31559 to bp 35164) into pNEB193, generating pNEBAd24E4. PNEBAd24E4 was then digested with AccI and EcoNI to remove the E4 coding sequences and ligated with an oligo designed to contain BglII and XhoI sites (underlined) (5′ ACTCGAGATGTATAGATCT (SEQ ID NO: 6); 5′CTAGATCTATACATCTCGAG (SEQ ID NO: 7)), generating pNEBAd24ΔE4. PNEBAd24ΔE4 was then digested with BglII and XhoI and ligated with the Ad5 Orf6 gene, which was PCR amplified, generating pNEBAd24ΔE4Ad5Orf6. The PCR primers used to amplify the Ad5 Orf6 gene (5′ GCACAGATCTTTGCTTCAGGAATATG (SEQ ID NO: 8); 5′ GAGAACTCGAGGCCTACATGGGGGTAGAG (SEQ ID NO: 9)) were designed to contain BglII and XhoI sites (underlined above) for ligation with the pNEBAd24DE4 fragment. In the final step pNEBAd24ΔE4Ad5Orf6 E4 shuttle plasmid was digested with PvuI and PmeI, the restriction fragments were size fractionated by agarose gel electrophoresis and the desired fragment containing Ad5Orf6 flanked by Ad24 sequences was gel purified. Cotransformation of BJ 5183 bacteria with E4 shuttle fragment and pAd24ΔOrf6BstZ17I, which had been linearized in the E4 region by digestion with BstZ17I, resulted in the generation of pAd24ΔE1ΔE4Ad5Orf6 by homologous recombination. Potential clones were screened by restriction analysis and one clone was selected as pre-adenovirus plasmid pAd24ΔE1ΔE4Ad5Orf6.

[0117] To construct pAd24ΔE1ΔOrf6Ad5Orf6 (An Ad24 pre-Ad plasmid containing an E1 deletion and a deletion of E4 Orf6 substituted with Ad5 Orf6), an Ad24 E4 shuttle plasmid was constructed in which the Ad24 Orf6 gene was replaced by Ad5 Orf6. To do this the EcoRI restriction fragment representing bp 32126 to bp 33442 of the Ad24 genome (encompassing the E4 Orf6 coding region), was subcloned into the EcoRI site in pNEB193, generating pNEBAd24Orf6. In order to delete the E4 Orf6 gene in pNEBAd24Orf6 and replace it with Ad5 Orf6, pNEBAd24Orf6 was digested with StyI and treated with Klenow to blunt the ends and then digested with to EagI. The desired pNEBAd24Orf6 fragment was then ligated with a PCR product representing the Ad5 Orf6 gene from Ad5 bp 33193 to bp 24125, generating pNEBAd24ΔOrf6Ad5Orf6. The PCR primers used to generate the Ad5 Orf6 fragment (5′CGAGACGGCCGACGCAGATCTGTTTG (SEQ ID NO: 10); 5′GAAGTCCCGGGCTACATGGGGGTAG (SEQ ID NO: 11)) were designed to contain EagI and SmaI sites (underlined above) for ligation with the pNEBAd24Orf6 fragment. In the final step pNEBAd24ΔOrf6Ad5Orf6 was digested with EcoRI, the restriction fragments were size fractionated by agarose gel electrophoresis and the desired fragment containing Ad5Orf6 flanked by Ad24 sequences was gel purified. Cotransformation of BJ 5183 bacteria with the EcoRI fragment and pAd24ΔOrf6BstZ17I, which had been linearized in the E4 region by digestion with BstZ17I, resulted in the generation of pAd24ΔE1ΔOrf6Ad5Orf6 by homologous recombination. Potential clones were screened by restriction analysis and one clone was selected as pre-adenovirus plasmid pAd24ΔE1ΔOrf6Ad5Orf6.

EXAMPLE 10

[0118] Rescue of pAd24ΔE1ΔE4Ad5Orf6, pAd24ΔE1ΔOrf6Ad5Orf6, into Virus

[0119] In order to determine if pre-adenovirus plasmids pAd24ΔE1ΔE4Ad5Orf6, pAd24ΔE1ΔOrf6Ad5Orf6, could be rescued into virus and propagated in a group C E1 complementing cell line, the plasmids were each digested with Pme I and transfected into T-25 flasks of PER.C6 cells using the calcium phosphate co-precipitation technique; (Cell Phect Transfection Kit, Amersham Pharmacia Biotech Inc.). PmeI digestion releases the viral genome from plasmid sequences allowing viral replication to occur after cell entry. Viral cytopathic effect (CPE), indicating that virus replication and amplification was occurring, was observed for both constructs. When CPE was complete, approximately 7-10 days post transfection, the infected cells and media were harvested, freeze/thawed three times and the cell debris pelleted by centrifugation. Approximately 1 ml of the cell lysate was used to infect T-225 flasks of PER.C6 cells at 80-90% confluence. Once CPE was reached, infected cells and media were harvested, freeze/thawed three times and the cell debris pelleted by centrifugation. Clarified cell lysates were then used to infect 2-layer NUNC cell factories of PER.C6 cells. Following complete CPE the virus was purified by ultracentrifugation on CsCl density gradients. In order to verify the genetic structure of the rescued viruses, viral DNA was extracted using pronase treatment followed by phenol chloroform extraction and ethanol precipitation. Viral DNA was then digested with HindIII and treated with Klenow fragment to end-label the restriction fragments with P33-dATP. The end-labeled restriction fragments were then size-fractionated by gel electrophoresis and visualized by autoradiography. The digestion products were compared with the digestion products of the corresponding pre-Adenovirus plasmid (that had been digested with Pme1/HindIII prior to labeling) from which they were derived. The expected sizes were observed, indicating that the viruses had been successfully rescued.

EXAMPLE 11

[0120] Comparison of the Growth Kinetics of Ad24 Based Vectors.

[0121] In order to compare the growth kinetic of Ad24ΔE1, Ad24ΔE1Ad5Orf6, Ad24ΔE1ΔE4Ad5Orf6 and Ad24ΔE1ΔOrf6Ad5Orf6 one step growth curves were preformed (FIG. 15). PER.C6 cells in 60 mm dishes were infected at 1 vp per cell with either Ad24ΔE1, Ad24ΔE1Ad5Orf6, Ad24ΔE1ΔE4Ad5Orf6 or Ad24ΔE1ΔOrf6Ad5Orf6. Cells and media were then harvested at various times post infection, freeze thawed three times and clarified by centrifugation. The amount of virus present in the samples was determined by quantitative PCR and is illustrated in FIG. 15. This study demonstrates that Ad24 vectors that incorporate Ad5 Orf6 have a distinct growth advantage over Ad24ΔE1 in PER.C6 cells. The instant invention can be practiced with recombinant Ad24 vectors absent a heterologous Orf 6 region where the E1-complementing cell line expresses an Ad24 μl region or, alternatively, E1 and E4 regions of the same serotype (such as Ad5E1/E4-expressing cell lines).

EXAMPLE 12

[0122] Insertion of an Expression Cassette into pAd24ΔE1ΔE4Ad5Orf6, pAd24ΔE1ΔOrf6Ad5Orf6.

[0123] In order to introduce a gag or SEAP expression cassette (see FIGS. 6 and 7, respectively) into the E1 region of the Ad24 pre-Adenovirus plasmids described above (pAd24ΔE1ΔE4Ad5Orf6, pAd24ΔE1ΔOrf6Ad5Orf6) bacterial recombination was used. A gag expression cassette consisting of the following: 1) the immediate early gene promoter from the human cytomegalovirus, 2) the coding sequence of the human immunodeficiency virus type 1 (HIV-1) gag (strain CAM-1; 1526 bp) gene, and 3) the bovine growth hormone polyadenylation signal sequence, was cloned into the E1 deletion in Ad17 shuttle plasmid, pABSAd17-3, generating pABSAd17HCMVgagBGHpA. The ITR cassette contains sequences from the right (bp 34469 to 35098) and left (bp 4 to 414 and bp 3373 to 4580) end of the Ad17 genome separated by plasmid sequences containing a bacterial origin of replication and an Ampicillin resistance gene. The ITR cassette contains a deletion of E1 sequences from Ad17 (bp 415 to 3372) with a unique Swa I site located in the deletion. The gag expression cassette was obtained from a previously constructed shuttle plasmid by EcoRI digestion. Following the digestion the desired fragment was gel purified, treated with Klenow to obtain blunt ends and cloned into the SwaI site in pABSAd17-3. This cloning step resulted in the gag expression cassette being cloned into the E1 deletion between bp 414 and 3373 in the E1 parallel orientation. The shuttle vector containing the gag transgene was digested to generate a DNA fragment consisting of the gag expression cassette flanked by Ad17 bp 4 to 414 and bp 3373 to 4580 and the fragment was purified after electrophoresis on an agarose gel. Cotransformation of BJ 5183 bacteria with the shuttle vector fragment and one of the Ad24 pre-Ad plasmids (pAd24ΔE1ΔE4Ad5Orf6, pAd24ΔE1ΔOrf6Ad5Orf6,), linearized in the E1 region by digestion with Swa I, resulted in the generation of the corresponding Ad24 gag-containing pre-Adenovirus plasmids (pAd24ΔE1gagΔE4Ad5Orf6, pAd24ΔE1gagΔOrf6Ad5Orf6) by homologous recombination. Potential clones were screened by restriction analysis.

[0124] A similar strategy was used to generate Ad24 pre-Ad plasmids containing a SEAP expression cassette. In this case a SEAP expression cassette consisting of: 1) the immediate early gene promoter from the human cytomegalovirus, 2) the coding sequence of the human placental SEAP gene, and 3) the bovine growth hormone polyadenylation signal sequence was cloned into the E1 deletion in Ad17 shuttle plasmid, pABSAd17-3, generating pABSAd17HCMVSEAPBGH. The SEAP expression cassette was obtained from a previously constructed shuttle plasmid by EcoRI digestion. Following the digestion the desired fragment was gel purified, treated with Klenow to obtain blunt ends and cloned into the SwaI site in pABSAd17-3. The shuttle vector containing the SEAP transgene was digested to generate a DNA fragment consisting of the SEAP expression cassette flanked by Ad17 bp 4 to 414 and bp 3373 to 4580 and the fragment was purified after electrophoresis on an agarose gel. Cotransformation of BJ 5183 bacteria with the shuttle vector fragment and one of the Ad24 pre-Ad plasmids (pAd24ΔE1ΔE4Ad5Orf6, pAd24ΔE1ΔOrf6Ad5Orf6,), linearized in the E1 region by digestion with Swa I, resulted in the generation of the corresponding Ad24 SEAP-containing pre-Adenovirus plasmids (pAd24ΔE1SEAPΔE4Ad5Orf6, pAd24ΔE1 SEAPΔOrf6Ad5Orf6) by homologous recombination. Potential clones were screened by restriction analysis. All pre-Ad plasmids were rescued into virus and expanded to prepare CsCl purified stocks as described above.

EXAMPLE 13

[0125] In Vivo Immunogenicity

[0126] A. Immunization

[0127] Cohorts of 3-6 animals were given intramuscular injections at wk 0 and wk 4 of either of the following constructs: (1) 10{circumflex over ( )}11 vp MRKAd5-HIV1 gag; (2) 10{circumflex over ( )}10 vp MRKAd5-HIV1 gag; (3) 10{circumflex over ( )}11 vp of Ad24ΔE1gagΔOrf6Ad5Orf6; (4) 10{circumflex over ( )}10 vp of Ad24ΔE1gagΔOrf6Ad5Orf6; or (5) 10{circumflex over ( )}10 vp of Ad24ΔE1gagΔE4Ad5Orf6. Rhesus macaques were between 3-10 kg in weight. In all cases, the total dose of each vaccine was suspended in 1 mL of buffer. The macaques were anesthetized (ketamine/xylazine) and the vaccines were delivered i.m. in 0.5-mL aliquots into both deltoid muscles using tuberculin syringes (Becton-Dickinson, Franklin Lakes, N.J.). Peripheral blood mononuclear cells (PBMC) were prepared from blood samples collected at several time points (typically 4 wk intervals) during the immunization regimen. All animal care and treatment were in accordance with standards approved by the Institutional Animal Care and Use Committee according to the principles set forth in the Guide for Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council.

[0128] B. ELISPOT Assay

[0129] The IFN-γ ELISPOT assays for rhesus macaques were conducted following a previously described protocol (Allen et al., 2001 J. Virol. 75(2):738-749; Casimiro et al., 2002 J. Virol. 76:185-94), with some modifications. For antigen-specific stimulation, a peptide pool was prepared from 20-aa peptides that encompass the entire HIV-1 gag sequence with 10-aa overlaps (Synpep Corp., Dublin, Calif.). To each well, 50 μL of 2-4×10⁵ peripheral blood mononuclear cells (PBMCs) were added; the cells were counted using Beckman Coulter Z2 particle analyzer with a lower size cut-off set at 80 femtoliters (“fL”). Either 50 μL of media or the gag peptide pool at 8 μg/mL concentration per peptide was added to the PBMC. The samples were incubated at 37° C., 5% CO₂ for 20-24 hrs. Spots were developed accordingly and the plates were processed using custom-built imager and automatic counting subroutine based on the ImagePro platform (Silver Spring, Md.); the counts were normalized to 10⁶ cell input.

[0130] C. Intracellular Cytokine Staining

[0131] To 1 ml of 2×10⁶ PBMC/mL in complete RPMI media (in 17×100 mm round bottom polypropylene tubes (Sarstedt, Newton, N.C.)), anti-hCD28 (clone L293, Becton-Dickinson) and anti-hCD49d (clone L25, Becton-Dickinson) monoclonal antibodies were added to a final concentration of 1 μg/mL. For gag-specific stimulation, 10 μL of the peptide pool (at 0.4 mg/mL per peptide) were added. The tubes were incubated at 37° C. for 1 hr., after which 20 μL of 5 mg/mL of brefeldin A (Sigma) were added. The cells were incubated for 16 hr at 37° C., 5% CO₂, 90% humidity. 4 mL cold PBS/2% FBS were added to each tube and the cells were pelleted for 10 min at 1200 rpm. The cells were re-suspended in PBS/2% FBS and stained (30 min, 4° C.) for surface markers using several fluorescent-tagged mAbs: 20 μL per tube anti-hCD3-APC, clone FN-18 (Biosource); 20 μL anti-hCD8-PerCP, clone SK1 (Becton Dickinson); and 20 μL anti-hCD4-PE, clone SK3 (Becton Dickinson). Sample handling from this stage was conducted in the dark. The cells were washed and incubated in 750 μL 1×FACS Perm buffer (Becton Dickinson) for 10 min at room temperature. The cells were pelleted and re-suspended in PBS/2% FBS and 0.1 μg of FITC-anti-hIFN-γ, clone MD-1 (Biosource) was added. After 30 min incubation, the cells were washed and re-suspended in PBS. Samples were analyzed using all four color channels of the Becton Dickinson FACSCalibur instrument. To analyze the data, the low side- and forward-scatter lymphocyte population was initially gated; a common fluorescence cut-off for cytokine-positive events was used for both CD4⁺ and CD8⁺ populations, and for both mock and gag-peptide reaction tubes of a sample.

[0132] D. Anti-p24 ELISA

[0133] A modified competitive anti-p24 assay was developed using reagents from the Coulter p24 Antigen Assay kit (Beckman Coulter, Fullerton, Calif.). Briefly, to a 250-μL serum sample, 20 μL of Lyse Buffer and 15 μL of p24 antigen (9.375 pg) from the Coulter kit were added. After mixing, 200 μL of each sample were added to wells coated with a mouse anti-p24 mAb from the Coulter kit and incubated for 1.5 hr at 37° C. The wells were then washed and 200 μL of Biotin Reagent (polyclonal anti-p24-biotin) from the Coulter kit was added to each well. After a 1 hr, 37° C. incubation, detection was achieved using strepavidin-conjugated horseradish peroxidase and TMB substrate as described in the Coulter Kit. OD_(450nm) values were recorded. A 7-point standard curve was generated using a serial 2-fold dilution of serum from an HIV-seropositive individual. The lower cut-off for the assay is arbitrarily set at 10 milli Merck units/mL (mMU/mL) defined by a dilution of the seropositive human serum. This cutoff falls at approximately 65% of the maximum bound control signal which corresponds to that obtained with the diluent control only and with no positive analyte.

[0134] E. Results

[0135] PBMCs collected at regular 4-wk intervals were analyzed in an ELISPOT assay (FIG. 17). Both Ad24ΔE1gagΔOrf6Ad5Orf6 and Ad24ΔE1gagΔE4Ad5Orf6 were able to induce significant levels of gag-specific T cells in non-human primates. At 10{circumflex over ( )}11 vp dose level, the Ad24-induced responses were within 2-3-fold of those of MRKAd5-HIV1 gag. Both Ad24 vectors were also able to induce detectable levels of gag-specific T cells at 10{circumflex over ( )}10 vp but were lower than those observed using MRKad5gag at the same dose.

[0136] PBMCs collected at wk 12 from the vaccinees were analyzed for intracellular IFN-γ staining after the priming immunizations. The assay results provided information on the relative amounts of CD4⁺ and CD8⁺ gag-specific T cells in the peripheral blood (FIG. 18). The results indicated that the prime-boost immunization approach was able to elicit in rhesus macaques both HIV-specific CD4⁺ and CD8⁺ T cells.

[0137] F. Humoral Immune Responses

[0138] The Ad24-based vaccine vector was able to generate detectable levels of circulating anti-gag antibodies at the reasonably high dose level (FIG. 19). No detectable titers were observed at equal to or lower than 10{circumflex over ( )}10 vp, suggesting the existence of a dose-dependent response.

EXAMPLE 14

[0139] In Vivo Transgene Expression

[0140] A. Immunization

[0141] Cohorts of 5 C3H/HeN mice were given single intramuscular injections of one of the following vectors: (1) 10{circumflex over ( )}10 vp Ad24ΔE1SEAPΔE4Ad5Orf6; (2) 10{circumflex over ( )}10 vp Ad24ΔE1SEAPΔOrf6Ad5Orf6; (3) 10{circumflex over ( )}10 vp MRKAd5SEAP; and (4) 10{circumflex over ( )}9 vp MRKAd5SEAP. Female mice were between 4-10 weeks old. The total dose of each vaccine was suspended in 0.1 mL of buffer. The vectors were given to both quadriceps of each of the animals with a volume of 50 uL per quad and using 0.3-mL 28G1/2 insulin syringes (Becton-Dickinson, Franklin Lakes, N.J.). For the primates, the total dose of each vaccine was suspended in 1 mL of buffer. The monkeys were anesthetized (ketamine/xylazine mixture) and the vaccines were delivered i.m. in 0.5-mL aliquots into two muscle sites using tuberculin syringes (Becton-Dickinson, Franklin Lakes, N.J.). Serum samples were collected at defined intervals and stored frozen until the assay date. All animal care and treatment were in accordance with standards approved by the Institutional Animal Care and Use Committee according to the principles set forth in the Guide for Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council.

[0142] B. SEAP Assay

[0143] Serum samples were analyzed for circulating SEAP levels using TROPIX phospha-light chemiluminescent kit (Applied Biosystems Inc). Duplicate 5 uL aliquots of each serum were mixed with 45 uL of kit-supplied dilution buffer in a 96-well white DYNEX plate. Serially diluted solutions of a human placental alkaline phosphatase (Catalog no. M5905, Sigma, St. Louis, Mo.) in 10% naive monkey serum served to provide the standard curve. Endogenous SEAP activity in the samples was inactivated by heating the wells for 30 minutes at 65° C. Enzymatic SEAP activities in the samples were determined following the procedures described in the kit. Chemiluminescence readings (in relative light units) were recorder using DYNEX luminometer. RLU readings are converted to ng/mL SEAP using a log-log regression analyses.

[0144] C. Rodent Results

[0145] Serum samples prior to and after the injection were analyzed for circulating SEAP activities and the results are shown in FIG. 20. Results indicate that (1) both Ad24 constructs are all capable of expressing the SEAP transgene in vivo to comparable levels; and that (2) the level of expression achieved using the Ad24 vectors are comparable to that of Ad5 at 10-fold lower dose. The levels of SEAP in the serum dropped dramatically after day 2 and were at background levels by day 12.

[0146] D. Primate Results

[0147] Cohorts of 3 rhesus macaques were given single intramuscular injections of one of the following vectors: (1) 10{circumflex over ( )}11 vp MRKAd5-SEAP; (2) 10{circumflex over ( )}9 vp MRKAd5-SEAP; (3) 10{circumflex over ( )}11 vp Ad24ΔE1SEAPΔOrf6Ad5Orf6; or (4) 10{circumflex over ( )}11 vp Ad24ΔE1SEAPΔE4Ad5Orf6. Serum samples prior to and after the injection were analyzed for circulating SEAP activities and the results are shown in FIG. 21.

[0148] Results indicate that the peak levels of SEAP product produced by adenovirus serotype 24 were lower than but were within 3-fold of that of MRKAd5 at the same high dose level of 10{circumflex over ( )}11 vp (FIG. 21). The levels observed with adenovirus serotype 24 are generally 50-fold higher than those observed using 10{circumflex over ( )}9 vp of MRKAd5. The levels of SEAP in the serum dropped dramatically after day 10 and were close to background as early as day 15. These observations strongly indicate that adenovirus serotype 24 is very efficient in expressing a transgene following intramuscular administration in a primate.

EXAMPLE 15

[0149] Construction of pMRKAd24ΔE1ΔE4Ad5Orf6

[0150] To construct pMRKAd24ΔE1ΔE4Ad5Orf6 (An Ad24 pre-Ad plasmid, composed entirely of Ad24 sequence and containing an E1 deletion and an E4 deletion substituted with Ad5 Orf6), an Ad24 ITR cassette was constructed containing sequences from the right (bp 31978 to 32264 and bp 34713 to 35164) and left (bp 4 to 450 and bp 3364 to 3799) end of the Ad24 genome separated by plasmid sequences containing a bacterial origin of replication and an ampicillin resistance gene. These four segments were generated by PCR and cloned sequentially into pNEB193, generating pNEBAd24-4. Next the Ad5 Orf6 open reading frame (Ad5 bp 31192 to bp 34078) was generated by PCR and cloned between Ad24 bp 32264 and 34713 generating pNEBAd24E-Ad5Orf6 (the ITR cassette). PNEB193 is a commonly used commercially available cloning plasmid (New England Biolabs cat# N3051S) containing a bacterial origin of replication, ampicillin resistance gene and a multiple cloning site into which the PCR products were introduced. The ITR cassette contains a deletion of E1 sequences from Ad24 bp 451 to 3363 with a unique Swa I restriction site located in the deletion and an E4 deletion from Ad24 bp 32265 to 34712 into which Ad5 Orf6 was introduced in an E4 parallel orientation. In this construct Ad5 Orf6 expression is driven by the Ad24 E4 promoter. The Ad24 sequences (bp 31978 to 32264 and bp 3464 to 3799) in the ITR cassette provide regions of homology with the purified Ad24 viral DNA in which bacterial recombination can occur following cotransformation into BJ 5183 bacteria (FIG. 22). The ITR cassette was also designed to contain unique restriction enzyme sites (PmeI) located at the end of the viral ITR's so that digestion will release the recombinant Ad24 genome from plasmid sequences. Potential clones will be screened by restriction analysis and one clone was selected as pMRKAd24ΔE1ΔE4Ad5Orf6. Pre-Adenovirus plasmid pMRKAd24ΔE1ΔE4Ad5Orf6 should contain Ad24 sequences from bp 4 to 450; bp 3364 to bp 32264 and bp 34713 to bp 35164 with Ad5Orf6 cloned between bp 32264 and bp 34713. The bp numbering in the above description refers to the wt sequence for both Ad24 and Ad5.

EXAMPLE 16

[0151] Insertion of HIV-1 gag and SEAP Transgenes into pAd24ΔE1ΔE4Ad5Orf6

[0152] In order to introduce a gag or SEAP expression cassettes into the E1 region of pMRKAd24ΔE1ΔE4Ad5Orf6, bacterial recombination will be used. An HIV-1 gag expression cassette will consist of the following: 1) the immediate early gene promoter from the human cytomegalovirus, 2) the coding sequence of the human immunodeficiency virus type 1 (HIV-1) gag (strain CAM-1; 1526 bp) gene, and 3) the bovine growth hormone polyadenylation signal sequence, in the E1 deletion of an Ad24 shuttle plasmid, pNEBAd24-2 (a precursor to the Ad24 ITR cassette described above), generating pNEBAd24CMVgagBGHpA. PNEBAd24-2 contains Ad24 sequences from the left end of the genome (bp 4 to 450 and bp 3364 to 3799) that define the E1 deletion. The gag expression cassette will be obtained from a previously constructed plasmid and cloned into the E1 deletion between bp 450 and 3364 in the E1 parallel orientation. The shuttle vector containing the gag transgene will be digested to generate a DNA fragment consisting of the gag expression cassette flanked by Ad24 bp 4 to 450 and bp 3364 to 3799 and the fragment will be purified after electrophoresis on an agarose gel. Cotransformation of BJ 5183 bacteria with the shuttle vector fragment and pMRKAd24ΔE1ΔE4Ad5Orf6 which was linearized in the E1 region by digestion with SwaI, should result in the generation of Ad24 gag-containing pre-Adenovirus plasmids pMRKAd24ΔE1gagΔE4Ad5Orf6 by homologous recombination. Potential clones will be screened by restriction analysis.

[0153] A similar strategy will be used to generate Ad24 pre-Ad plasmids containing a SEAP expression cassette. In this case, a SEAP expression cassette will consist of: 1) the immediate early gene promoter from the human cytomegalovirus, 2) the coding sequence of the human placental SEAP gene, and 3) the bovine growth hormone polyadenylation signal sequence cloned into the E1 deletion of an Ad24 shuttle plasmid, pNEBAd24-2, generating pNEBAd24CMVSEAPBGHpA. The transgene will then be recombined into pMRKAd24ΔE1ΔE4Ad5Orf6 as described above for the gag transgene.

EXAMPLE 17

[0154] In Vivo Immunogenicity

[0155] A. Immunization

[0156] Rhesus macaques were between 3-10 kg in weight. In all cases, the total dose of each vaccine was suspended in 1 mL of buffer. The macaques were anesthetized (ketamine/xylazine) and the vaccines were delivered i.m. in 0.5-mL aliquots into both deltoid muscles using tuberculin syringes (Becton-Dickinson, Franklin Lakes, N.J.). Peripheral blood mononuclear cells (PBMC) were prepared from blood samples collected at several time points during the immunization regimen. All animal care and treatment were in accordance with standards approved by the Institutional Animal Care and Use Committee according to the principles set forth in the Guide for Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council.

[0157] B. T Cell Responses

[0158] Ad24 Vaccine Vector as a Heterologous Booster: Cohort of 4 rhesus macaques was immunized initially with 3 doses (wk 0, 4, 26) of either 10⁷ or 10⁹ vp of MRKAd5-gag (see, PCT/US01/28861, published Mar. 21, 2002) or MRKAd6-gag. At wk 56, the animals received a booster vaccine of 10¹¹ vp Ad24ΔE1gagΔOrf6Ad5Orf6. A separate cohort of naïve animals received a single dose of the booster vaccine. The results of the IFN-γ ELISPOT analyses of PBMC collected during the course of the studies are shown in FIG. 23. It is apparent that the Ad24 HIV vectors can be utilized to amplify the existing pools of HIV-specific T cells. The increases in the levels of gag-specific T cells from the pre-boost levels to those measured at 4 wks post boost were consistently larger than the levels induced by the same booster vaccine in naive animals. PBMCs from the vaccinees of the heterologous MRKAd5/MRKAd6-Ad24 boost regimen were analyzed for intracellular IFN-γ staining after the priming immunizations (wk 60). The assay results provided information on the relative amounts of CD4⁺ and CD8⁺ gag-specific T cells in the peripheral blood (FIG. 24). The results indicated that heterologous prime-boost immunization approach was able to elicit in rhesus macaques both HIV-specific CD4+ and CD8+ T cells.

[0159] Ad24 Vaccine Vector as a Heterologous Primer: In a separate study, a cohort of 3 rhesus macaques was immunized initially with 2 doses (wk 0, 4) of 10¹¹ vp Ad24ΔE1gagΔOrf6Ad5Orf6 and boosted at wk 24 with 10⁷ vp of MRKAd5-gag. The low dose of MRKAd5-gag is selected to mimic the effect of pre-existing neutralizing immunity to the vector in a subject. A separate cohort of naïve animals was given a single dose of 10⁷ vp MRKAd5-gag. The results of the IFN-γ ELISPOT analyses of PBMC collected during the course of the studies are shown in FIG. 25.

[0160] The Ad24-based vaccine was able to prime effectively for HIV-specific T cell responses in macaques. Boosting with a low dose MRKAd5-gag resulted in a significant increase in the levels of gag-specific T cells. The increases in 2 out of 3 animals exceed the levels typically observed after treatment of naïve animals with the same low dose of MRKAd5-gag.

EXAMPLE 18

[0161] Construction of pAd34ΔE1ΔE4Ad5Orf6

[0162] To generate an E1-Ad34 based vector that can propagate in existing group C/Ad5 E1 complementing cell lines (293, PER.C6), Ad5 Orf6 was inserted in place of the native E4 region. Since at the time, the complete sequence of Ad34 (see FIGS. 28A-1 to 28A-9; subject of copending application serial No. 60/458,825, filed Mar. 28, 2003) was unknown, advantage was taken of the sequence homology between Ad34 and Ad35 in order to construct the Ad34 pre-Adenovirus plasmid. Cotransformation of BJ 5183 bacteria with purified wild-type Ad34 viral DNA and the appropriately constructed Ad35 ITR cassette resulted in the circularization of the viral genome by homologous recombination. The construction of the pre-Ad plasmid based on Ad34, is outlined below:

[0163] To construct pAd34ΔE1ΔE4Ad5Orf6 (An Ad34 pre-Ad plasmid containing an E1 deletion and an E4 deletion substituted with Ad5 Orf6), we utilized an Ad35 ITR cassette. We anticipated that sequence homology between Ad34 and Ad35 would allow homologous recombination to occur. The Ad35 ITR cassette was constructed containing sequences from the right (bp 31599 to 31913 and bp 34419 to 34793) and left (bp 4 to 456 and bp 3403 to 3886) end of the Ad35 genome (see FIGS. 2A-1 to 2A-10) separated by plasmid sequences containing a bacterial origin of replication and an ampicillin resistance gene. The four segments were generated by PCR and cloned sequentially into pNEB193, generating pNEBAd35-4. Next the Ad5 Orf6 open reading frame was generated by PCR and cloned between Ad35 bp 31913 and 34419 generating pNEBAd35-4Ad5Orf6 (the ITR cassette). PNEB193 is a commonly used commercially available cloning plasmid (New England Biolabs cat# N3051S) containing a bacterial origin of replication, ampicillin resistance gene and a multiple cloning site into which the PCR products were introduced. The ITR cassette contains a deletion of E1 sequences from Ad35 bp 457 to 3402 with a unique Swa I restriction site located in the deletion and an E4 deletion from Ad35 bp 31914 to 34418 into which Ad5 Orf6 was introduced in an E4 parallel orientation. In this construct Ad5Orf6 expression is driven by the Ad35 E4 promoter. The Ad35 sequences (bp 31599 to 31913 and bp 3403 to 3886) in the ITR cassette provided regions of homology with the purified Ad34 viral DNA in which bacterial recombination could occur following cotransformation into BJ 5183 bacteria (FIG. 26). The ITR cassette was also designed to contain unique restriction enzyme sites (PmeI) located at the end of the viral ITR's so that digestion would release the recombinant Ad34 genome from the plasmid sequences. Potential clones were screened by restriction analysis and one clone was selected as pAd34ΔE1ΔE4Ad5Orf6.

EXAMPLE 19

[0164] Rescue of pAd34ΔE1ΔE4Ad5Orf6 into Virus

[0165] In order to determine if pre-adenovirus plasmid pAd34ΔE1ΔE4Ad5Orf6, could be rescued into virus and propagated in a group C E1 complementing cell line, the plasmid was digested with Pme I and transfected into T-25 flasks of PER.C6 cells using the calcium phosphate co-precipitation technique (Cell Phect Transfection Kit, Amersham Pharmacia Biotech Inc). PmeI digestion releases the viral genome from plasmid sequences allowing viral replication to occur after cell entry. Viral cytopathic effect (CPE), indicating that virus replication and amplification was occurring was observed following transfection. When CPE was complete, approximately 7-10 days post transfection, the infected cells and media were harvested, freeze/thawed three times and the cell debris pelleted by centrifugation. Approximately 1 ml of the cell lysate was used to infect a T-225 flask of PER.C6 cells at 80-90% confluence. Once CPE was reached, infected cells and media were harvested, freeze/thawed three times and the cell debris pelleted by centrifugation. Clarified cell lysates were then used to infect 2-layer NUNC cell factories of PER.C6 cells. Following complete CPE, the virus was purified by ultracentrifugation on CsCl density gradients. In order to verify the genetic structure of the rescued viruses, viral DNA was extracted using pronase treatment followed by phenol chloroform extraction and ethanol precipitation. Viral DNA was then digested with HindIII and treated with Klenow fragment to end-label the restriction fragments with P33-dATP. The end-labeled restriction fragments were then size-fractionated by gel electrophoresis and visualized by autoradiography. The digestion products were compared with the digestion products of the corresponding pre-Adenovirus plasmid (that had been digested with Pme1/HindIII prior to labeling) from which they were derived. The expected sizes were observed, indicating that the viruses had been successfully rescued.

EXAMPLE 20

[0166] Insertion of an Expression Cassette into pAd34ΔE1ΔE4Ad5Orf6

[0167] In order to introduce a gag or SEAP expression cassette (see FIGS. 6 and 7, respectively) into the E1 region of pAd34ΔE1ΔE4Ad5Orf6, bacterial recombination was again used. A gag expression cassette consisting of the following: 1) the immediate early gene promoter from human cytomegalovirus, 2) the coding sequence of the human immunodeficiency virus type 1 (HIV-1) gag (strain CAM-1; 1526 bp) gene, and 3) the bovine growth hormone polyadenylation signal sequence, was cloned into the E1 deletion in Ad35 shuttle plasmid, pNEBAd35-2 (a precursor to the Ad35 ITR cassettes described above), generating pNEBAd35CMVgagBGHpA. pNEBAd35-2 contains Ad35 sequences from the left end of the genome (bp 4 to 456 and bp 3403 to 3886) with a unique SwaI site between bp 456 and 3403 at the position of the deletion. The gag expression cassette was obtained from a previously constructed shuttle plasmid by EcoRI digestion. Following the digestion the desired fragment was gel purified, treated with Klenow to obtain blunt ends and cloned into the SwaI site in pNEBAd35-2. This cloning step resulted in the gag expression cassette being inserted into the E1 deletion between bp 456 and 3403 in the E1 parallel orientation. The shuttle vector containing the gag transgene was digested to generate a DNA fragment consisting of the gag expression cassette flanked by Ad35 bp 4 to 456 and bp 3403 to 3886 and the fragment was purified after electrophoresis on an agarose gel. Cotransformation of BJ 5183 bacteria with the shuttle vector fragment and pAd34ΔE1ΔE4Ad5Orf6, linearized in the E1 region by digestion with Swa I, resulted in the generation of the Ad34 gag-containing pre-Adenovirus plasmid pAd34ΔE1gagΔE4Ad5Orf6 by homologous recombination. Potential clones were screened by restriction analysis.

[0168] A similar strategy was used to generate Ad34 pre-Ad plasmids containing a SEAP expression cassette. In this case a SEAP expression cassette consisting of: 1) the immediate early gene promoter from human cytomegalovirus, 2) the coding sequence of the human placental SEAP gene, and 3) the bovine growth hormone polyadenylation signal sequence was cloned into the E1 deletion in Ad35 shuttle plasmid, pNEBAd35-2, generating pNEBAd35CMVSEAPBGHpA. The SEAP expression cassette was obtained from a previously constructed shuttle plasmid by EcoRI digestion. Following the digestion the desired fragment was gel purified, treated with Klenow to obtain blunt ends and cloned into the SwaI site in pNEBAd35-2. The transgene was then recombined into the pAd34ΔE1ΔE4Ad5Orf6, generating pAd34ΔE1SEAPΔE4Ad5Orf6 as described above for the gag transgene.

[0169] All pre-Ad plasmids were rescued into virus and expanded to prepare CsCl purified stocks as described above.

EXAMPLE 21

[0170] Construction of pMRKAd34ΔE1ΔE4Ad5Orf6

[0171] To construct an Ad34 pre-Ad plasmid that was composed entirely of Ad34 sequences, an Ad34 ITR cassette was generated. The Ad34 ITR cassette was constructed containing sequences from the right (bp 31584 to 31895 and bp 34409 to 34772) and left (bp 4 to 456 and bp 3402 to 3885) end of the Ad34 genome (see FIGS. 28A-1 to 28A-9) separated by plasmid sequences containing a bacterial origin of replication and an ampicillin resistance gene. These four segments were generated by PCR and cloned sequentially into pNEB 193, generating pNEBAd34-4. Next the Ad5 Orf6 open reading frame was generated by PCR and cloned between Ad34 bp 31895 and 34409 generating pNEBAd34-4Ad5Orf6 (the ITR cassette). PNEB193 is a commonly used commercially available cloning plasmid (New England Biolabs cat# N3051S) containing a bacterial origin of replication, ampicillin resistance gene and a multiple cloning site into which the PCR products were introduced. The ITR cassette contains a deletion of E1 sequences from Ad34 bp 457 to 3401 with a unique Swa I restriction site located in the deletion and an E4 deletion from Ad34 bp 31896 to 34408 into which Ad5 Orf6 was introduced in an E4 parallel orientation. In this construct Ad5Orf6 expression is driven by the Ad34 E4 promoter. The Ad34 sequences (bp 31584 to 31895 and bp 3402 to 3885) in the ITR cassette provided regions of homology with the purified Ad34 viral DNA in which bacterial recombination could occur following cotransformation into BJ 5183 bacteria (FIG. 27). The ITR cassette was also designed to contain unique restriction enzyme sites (PmeI) located at the end of the viral ITR's so that digestion would release the recombinant Ad34 genome from the plasmid sequences. Potential clones were screened by restriction analysis and one clone was selected as pMRKAd34ΔE1ΔE4Ad5Orf6.

EXAMPLE 22

[0172] In Vivo Studies

[0173] A. Immunization

[0174] Cohorts of 3 rhesus macaques were given single intramuscular injections of one of the two vectors: (1) 10{circumflex over ( )}11 vp MRKAd5-SEAP (in MRKAd vector backbone disclosed in PCT/US01/28861, published Mar. 21, 2002); and (2) 10{circumflex over ( )}11 Vp Ad34ΔE1SEAPΔE4Ad5Orf6. Rhesus macaques were between 3-10 kg in weight. In all cases, the total dose of each vaccine was suspended in 1 mL of buffer. The macaques were anesthetized (ketamine/xylazine) and the vaccines were delivered i.m. in 0.5-mL aliquots into both deltoid muscles using tuberculin syringes (Becton-Dickinson, Franklin Lakes, N.J.). Peripheral blood mononuclear cells (PBMC) were prepared from blood samples collected at several time points during the immunization regimen. All animal care and treatment were in accordance with standards approved by the Institutional Animal Care and Use Committee according to the principles set forth in the Guide for Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council.

[0175] B. SEAP Assay

[0176] Serum samples were analyzed for circulating human secreted alkaline phosphatase (SEAP) levels using TROPIX phospha-light chemiluminescent kit (Applied Biosystems Inc). Duplicate 5 μL aliquots of each serum were mixed with 45 μL of kit-supplied dilution buffer in a 96-well white DYNEX plate. Serially diluted solutions of a human placental alkaline phosphatase (Catalog no. M5905, Sigma, St. Louis, Mo.) in 10% naïve monkey serum served to provide the standard curve. Endogenous SEAP activity in the samples was inactivated by heating the well for 30 minutes at 65° C. Enzymatic SEAP activities in the samples were determined following the procedures described in the kit. Chemiluminescence readings (in relative light units) were recorded using DYNEX luminometer. RLU readings were converted to ng/mL SEAP using a log-log regression analyses.

[0177] C. ELISPOT Assay

[0178] The IFN-γ ELISPOT assays for rhesus macaques were conducted following a previously described protocol (Allen et al., 2001 J. Virol. 75(2):738-749), with some modifications. For antigen-specific stimulation, a peptide pool was prepared from 20-aa peptides that encompass the entire HIV-1 gag sequence with 10-aa overlaps (Synpep Corp., Dublin, Calif.). To each well, 50 μL of 2-4×10⁵ peripheral blood mononuclear cells (PBMCs) were added; the cells were counted using Beckman Coulter Z2 particle analyzer with a lower size cut-off set at 80 femtoliters (“fL”). Either 50 μL of media or the gag peptide pool at 8 pg/mL concentration per peptide was added to the PBMC. The samples were incubated at 37° C., 5% CO₂ for 20-24 hrs. Spots were developed accordingly and the plates were processed using custom-built imager and automatic counting subroutine based on the ImagePro platform (Silver Spring, Md.); the counts were normalized to 10⁶ cell input.

[0179] D. Intracellular Cytokine Staining (ICS)

[0180] To 1 ml of 2×10⁶ PBMC/mL in complete RPMI media (in 17×100 mm round bottom polypropylene tubes (Sarstedt, Newton, N.C.)), anti-hCD28 (clone L293, Becton-Dickinson) and anti-hCD49d (clone L25, Becton-Dickinson) monoclonal antibodies were added to a final concentration of 1 μg/mL. For gag-specific stimulation, 10 μL of the peptide pool (at 0.4 mg/mL per peptide) were added. The tubes were incubated at 37° C. for 1 hr., after which 20 μL of 5 mg/mL of brefeldin A (Sigma) were added. The cells were incubated for 16 hr at 37° C., 5% CO₂, 90% humidity. 4 mL cold PBS/2% FBS were added to each tube and the cells were pelleted for 10 min at 1200 rpm. The cells were re-suspended in PBS/2% FBS and stained (30 min, 4° C.) for surface markers using several fluorescent-tagged mAbs: 20 μL per tube anti-hCD3-APC, clone FN-18 (Biosource); 20 μL anti-hCD8-PerCP, clone SK1 (Becton Dickinson); and 20 μL anti-hCD4-PE, clone SK3 (Becton Dickinson). Sample handling from this stage was conducted in the dark. The cells were washed and incubated in 750 μL 1×FACS Perm buffer (Becton Dickinson) for 10 min at room temperature. The cells were pelleted and re-suspended in PBS/2% FBS and 0.1 μg of FITC-anti-hIFN-γ, clone MD-1 (Biosource) was added. After 30 min incubation, the cells were washed and re-suspended in PBS. Samples were analyzed using all four color channels of the Becton Dickinson FACSCalibur instrument. To analyze the data, the low side- and forward-scatter lymphocyte population was initially gated; a common fluorescence cut-off for cytokine-positive events was used for both CD4+ and CD8⁺ populations, and for both mock and gag-peptide reaction tubes of a sample.

[0181] E. Results

[0182] Expression: Serum samples prior to and after the injection were analyzed for circulating SEAP activities and the results are shown in FIG. 29. Results indicate that the peak levels of SEAP protein produced by the alternative adenovirus serotype were lower than but were within 3-fold of that of MRKAd5 at the same high dose level of 10{circumflex over ( )}11 vp (FIG. 29). The levels of SEAP in the serum dropped dramatically after day 10 and were close to background as early as day 15. These observations strongly indicate that the Ad34-based vector is efficient in expressing a transgene following intramuscular administration in a primate.

[0183] Immunogenicity: Vaccine-induced T cell responses against HIV-1 gag were quantified using IFN-gamma ELISPOT assay against a pool of 20-aa peptides that encompassed the entire protein sequence. The results are shown in FIG. 30; they are expressed as the number of spot-forming cells (SFC) per million peripheral blood mononuclear cells (PBMCs) that responded to the peptide pool or the mock (no peptide) control.

[0184] Immunization with gag-expressing Ad34 vector induced detectable levels of circulating gag-specific T cells immediately after a single dose of the vector. The responses improved following a second dose given at wk 4. Overall, the responses to the Ad34-based vector were slightly lower than those induced by the same dose of MRKAd5-gag. The results strongly indicate the Ad34-based vector can prime effectively for HIV-specific T cell responses.

[0185] IFN-γ ICS analyses of the PBMC from the Ad34-immunized animals revealed that the vector can induce detectable levels of both CD4⁺ and CD8⁺ HIV-specific T cells (FIG. 31).

EXAMPLE 23

[0186] Heterologous Immunization

[0187] Cohorts of 3 monkeys were immunized (at wks 0, 4) with 10{circumflex over ( )}11 vp Ad34ΔE1gagΔE4Ad5Orf6 followed by a booster at week 24 with 10{circumflex over ( )}10 vp Ad35ΔE1gagΔE4Ad5Orf6. Vaccine-induced T cell responses against HIV-1 gag were quantified using IFN-gamma ELISPOT assay against a pool of 20-aa peptides that encompassed the entire protein sequence. The results are shown in FIG. 32; they are expressed as the number of spot-forming cells (SFC) per million peripheral blood mononuclear cells (PBMCs) that responded to the peptide pool or the mock (no peptide) control.

[0188] Immunization with gag-expressing Ad34 vector induced detectable levels of circulating gag-specific T cells that decreased to between 94-139 SFC/10{circumflex over ( )}6 PBMC at the time of the boost. Heterologous immunization with an Ad35-based HIV vector resulted in as much as a 3-fold increase in T cell responses.

[0189] IFN-γ ICS analyses of the PBMCs from the Ad34 primed/Ad35 boosted animals at week 28 revealed that the vector can induce detectable levels of both CD4⁺ and CD8⁺ HIV-specific T cells (FIG. 33).

1 12 1 34796 DNA adenovirus serotype 35 1 catcatcaat aatatacctt atagatggaa tggtgccaat atgtaaatga ggtgatttta 60 aaaagtgtgg gccgtgtggt gattggctgt ggggttaacg gttaaaaggg gcggcgcggc 120 cgtgggaaaa tgacgtttta tgggggtgga gtttttttgc aagttgtcgc gggaaatgtt 180 acgcataaaa aggcttcttt tctcacggaa ctacttagtt ttcccacggt atttaacagg 240 aaatgaggta gttttgaccg gatgcaagtg aaaattgctg attttcgcgc gaaaactgaa 300 tgaggaagtg tttttctgaa taatgtggta tttatggcag ggtggagtat ttgttcaggg 360 ccaggtagac tttgacccat tacgtggagg tttcgattac cgtgtttttt acctgaattt 420 ccgcgtaccg tgtcaaagtc ttctgttttt acgtaggtgt cagctgatcg ctagggtatt 480 tatacctcag ggtttgtgtc aagaggccac tcttgagtgc cagcgagaag agttttctcc 540 tctgcgccgg cagtttaata ataaaaaaat gagagatttg cgatttctgc ctcaggaaat 600 aatctctgct gagactggaa atgaaatatt ggagcttgtg gtgcacgccc tgatgggaga 660 cgatccggag ccacctgtgc agctttttga gcctcctacg cttcaggaac tgtatgattt 720 agaggtagag ggatcggagg attctaatga ggaagctgtg aatggctttt ttaccgattc 780 tatgctttta gctgctaatg aaggattaga attagatccg cctttggaca ctttcaatac 840 tccaggggtg attgtggaaa gcggtacagg tgtaagaaaa ttacctgatt tgagttccgt 900 ggactgtgat ttgcactgct atgaagacgg gtttcctccg agtgatgagg aggaccatga 960 aaaggagcag tccatgcaga ctgcagcggg tgagggagtg aaggctgcca atgttggttt 1020 tcagttggat tgcccggagc ttctggacat ggctgtaagt cttgtgaatt tcacaggaaa 1080 aatactggag taaaggaact gttatgttcg cttttgttat atgaaaaccc actgccactt 1140 tatttacagt aaagtgtgtt taagttaaaa tttaaaggaa tatgctgttt ttcacatgta 1200 tattgagtgt gagttttgtg cttcttatta taagtcctgt gtctgatgct gatgaatcac 1260 catctcctga ttctactacc tcacctcctg atattcaagc acctgttcct gtggacgtgc 1320 gcaagcccat tcctgtgaag cttaagcctg ggaaacgtcc agcagtggag aaacttgagg 1380 acttgttaca gggtggggac ggacctttgg acttgagtac acggaaacgt ccaagacaat 1440 aagtgttcca tatccgtgtt tacttaaggt gacgtcaata tttgtgtgag agtgcaatgt 1500 aataaaaata tgttaactgt tcactggttt ttattgcttt ttgggcgggg actcaggtat 1560 ataagtagaa gcagacctgt gtggttagct cataggagct ggctttcatc catggaggtt 1620 tgggccattt tggaagacct taggaagact aggcaactgt tagagagcgc ttcggacgga 1680 gtctccggtt tttggagatt ctggttcgct agtgaattag ctagggtagt ttttaggata 1740 aaacaggact ataaacaaga atttgaaaag ttgttggtag attgcccagg actttttgaa 1800 gctcttaatt tgggccatca ggttcacttt aaagaaaaag ttttatcagt tttagacttt 1860 tcaaccccag gtagaactgc tgctgctgtg gcttttctta cttttatatt agataaatgg 1920 atcccgcaga ctcatttcag caggggatac gttttggatt tcatagccac agcattgtgg 1980 agaacatgga aggttcgcaa gatgaggaca atcttaggtt actggccagt gcagcctttg 2040 ggtgtagcgg gaatcctgag gcatccaccg gtcatgccag cggttctgga ggaggaacag 2100 caagaggaca acccgagagc cggcctggac cctccagtgg aggaggcgga gtagctgact 2160 tgtctcctga actgcaacgg gtgcttactg gatctacgtc cactggacgg gataggggcg 2220 ttaagaggga gagggcatcc agtggtactg atgctagatc tgagttggct ttaagtttaa 2280 tgagtcgcag acgtcctgaa accatttggt ggcatgaggt tcagaaagag ggaagggatg 2340 aagtttctgt attgcaggag aaatattcac tggaacaggt gaaaacatgt tggttggagc 2400 cagaggatga ttgggaggtg gccattaaaa attatgccaa gatagctttg aggcctgata 2460 aacagtataa gatcagtaga cggattaata tccggaatgc ttgttacata tctggaaatg 2520 gggctgaggt ggtaatagat actcaagaca agacagttat tagatgctgc atgatggata 2580 tgtggcctgg agtagtcggt atggaagcag tcacttttgt aaatgttaag tttaggggag 2640 atggttataa tggaatagtg tttatggcca ataccaaact tatattgcat ggttgtagct 2700 tttttggttt caacaatacc tgtgtagatg cctggggaca ggttagtgta cgggggtgta 2760 gtttctatgc gtgttggatt gccacagctg gcagaaccaa gagtcaattg tctctgaaga 2820 aatgcatatt ccaaagatgt aacctgggca ttctgaatga aggcgaagca agggtccgtc 2880 actgcgcttc tacagatact ggatgtttta ttttaattaa gggaaatgcc agcgtaaagc 2940 ataacatgat ttgtggtgct tccgatgaga ggccttatca aatgctcact tgtgctggtg 3000 ggcattgtaa tatgctggct actgtgcata ttgtttccca tcaacgcaaa aaatggcctg 3060 tttttgatca caatgtgttg accaagtgca ccatgcatgc aggtgggcgt agaggaatgt 3120 ttatgcctta ccagtgtaac atgaatcatg tgaaagtgtt gttggaacca gatgcctttt 3180 ccagaatgag cctaacagga atctttgaca tgaacacgca aatctggaag atcctgaggt 3240 atgatgatac gagatcgagg gtgcgcgcat gcgaatgcgg aggcaagcat gccaggttcc 3300 agccggtgtg tgtagatgtg accgaagatc tcagaccgga tcatttggtt attgcccgca 3360 ctggagcaga gttcggatcc agtggagaag aaactgacta aggtgagtat tgggaaaact 3420 ttggggtggg attttcagat ggacagattg agtaaaaatt tgttttttct gtcttgcagc 3480 tgacatgagt ggaaatgctt cttttaaggg gggagtcttc agcccttatc tgacagggcg 3540 tctcccatcc tgggcaggag ttcgtcagaa tgttatggga tctactgtgg atggaagacc 3600 cgttcaaccc gccaattctt caacgctgac ctatgctact ttaagttctt cacctttgga 3660 cgcagctgca gccgctgccg ccgcctctgt cgccgctaac actgtgcttg gaatgggtta 3720 ctatggaagc atcgtggcta attccacttc ctctaataac ccttctacac tgactcagga 3780 caagttactt gtccttttgg cccagctgga ggctttgacc caacgtctgg gtgaactttc 3840 tcagcaggtg gccgagttgc gagtacaaac tgagtctgct gtcggcacgg caaagtctaa 3900 ataaaaaaaa ttccagaatc aatgaataaa taaacgagct tgttgttgat ttaaaatcaa 3960 gtgtttttat ttcatttttc gcgcacggta tgccctggac caccgatctc gatcattgag 4020 aactcggtgg attttttcca gaatcctata gaggtgggat tgaatgttta gatacatggg 4080 cattaggccg tctttggggt ggagatagct ccattgaagg gattcatgct ccggggtagt 4140 gttgtaaatc acccagtcat aacaaggtcg cagtgcatgg tgttgcacaa tatcttttag 4200 aagtaggctg attgccacag ataagccctt ggtgtaggtg tttacaaacc ggttgagctg 4260 ggaggggtgc attcgaggtg aaattatgtg cattttggat tggattttta agttggcaat 4320 attgccgcca agatcccgtc ttgggttcat gttatgaagg actaccaaga cggtgtatcc 4380 ggtacattta ggaaatttat cgtgcagctt ggatggaaaa gcgtggaaaa atttggagac 4440 acccttgtgt cctccgagat tttccatgca ctcatccatg ataatagcaa tggggccgtg 4500 ggcagcggcg cgggcaaaca cgttccgtgg gtctgacaca tcatagttat gttcctgagt 4560 taaatcatca taagccattt taatgaattt ggggcggagc gtaccagatt ggggtatgaa 4620 tgttccttcg ggccccggag catagttccc ctcacagatt tgcatttccc aagctttcag 4680 ttctgagggt ggaatcatgt ccacctgggg ggctatgaag aacaccgttt cgggggcggg 4740 ggtgattagt tgggatgata gcaagtttct gagcaattga gatttgccac atccggtggg 4800 gccataaata attccgatta caggttgcag gtggtagttt agggaacggc aactgccgtc 4860 ttctcgaagc aagggggcca cctcgttcat catttccctt acatgcatat tttcccgcac 4920 caaatccatt aggaggcgct ctcctcctag tgatagaagt tcttgtagtg aggaaaagtt 4980 tttcagcggt tttagaccgt cagccatggg cattttggaa agagtttgct gcaaaagttc 5040 tagtctgttc cacagttcag tgatgtgttc tatggcatct cgatccagca gacctcctcg 5100 tttcgcgggt ttggacggct cctggagtag ggtatgagac gatgggcgtc cagcgctgcc 5160 agggttcggt ccttccaggg tctcagtgtt cgagtcaggg ttgtttccgt cacagtgaag 5220 gggtgtgcgc ctgcttgggc gcttgccagg gtgcgcttca gactcattct gctggtggag 5280 aacttctgtc gcttggcgcc ctgtatgtcg gccaagtagc agtttaccat gagttcgtag 5340 ttgagcgcct cggctgcgtg gcctttggcg cggagcttac ctttggaagt tttcttgcat 5400 accgggcagt ataggcattt cagcgcatac agcttgggcg caaggaaaat ggattctggg 5460 gagtatgcat ccgcgccgca ggaggcgcaa acagtttcac attccaccag ccaggttaaa 5520 tccggttcat tggggtcaaa aacaagtttt ccgccatatt ttttgatgcg tttcttacct 5580 ttggtctcca taagttcgtg tcctcgttga gtgacaaaca ggctgtccgt atctccgtag 5640 actgatttta caggcctctt ctccagtgga gtgcctcggt cttcttcgta caggaactct 5700 gaccactctg atacaaaggc gcgcgtccag gccagcacaa aggaggctat gtgggagggg 5760 tagcgatcgt tgtcaaccag ggggtccacc ttttccaaag tatgcaaaca catgtcaccc 5820 tcttcaacat ccaggaatgt gattggcttg taggtgtatt tcacgtgacc tggggtcccc 5880 gctggggggg tataaaaggg ggcggttctt tgctcttcct cactgtcttc cggatcgctg 5940 tccaggaacg tcagctgttg gggtaggtat tccctctcga aggcgggcat gacctctgca 6000 ctcaggttgt cagtttctaa gaacgaggag gatttgatat tgacagtgcc ggttgagatg 6060 cctttcatga ggttttcgtc catttggtca gaaaacacaa tttttttatt gtcaagtttg 6120 gtggcaaatg atccatacag ggcgttggat aaaagtttgg caatggatcg catggtttgg 6180 ttcttttcct tgtccgcgcg ctctttggcg gcgatgttga gttggacata ctcgcgtgcc 6240 aggcacttcc attcggggaa gatagttgtt aattcatctg gcacgattct cacttgccac 6300 cctcgattat gcaaggtaat taaatccaca ctggtggcca cctcgcctcg aaggggttca 6360 ttggtccaac agagcctacc tcctttccta gaacagaaag ggggaagtgg gtctagcata 6420 agttcatcgg gagggtctgc atccatggta aagattcccg gaagtaaatc cttatcaaaa 6480 tagctgatgg gagtggggtc atctaaggcc atttgccatt ctcgagctgc cagtgcgcgc 6540 tcatatgggt taaggggact gccccagggc atgggatggg tgagagcaga ggcatacatg 6600 ccacagatgt catagacgta gatgggatcc tcaaagatgc ctatgtaggt tggatagcat 6660 cgcccccctc tgatacttgc tcgcacatag tcatatagtt catgtgatgg cgctagcagc 6720 cccggaccca agttggtgcg attgggtttt tctgttctgt agacgatctg gcgaaagatg 6780 gcgtgagaat tggaagagat ggtgggtctt tgaaaaatgt tgaaatgggc atgaggtaga 6840 cctacagagt ctctgacaaa gtgggcataa gattcttgaa gcttggttac cagttcggcg 6900 gtgacaagta cgtctagggc gcagtagtca agtgtttctt gaatgatgtc ataacctggt 6960 tggtttttct tttcccacag ttcgcggttg agaaggtatt cttcgcgatc cttccagtac 7020 tcttctagcg gaaacccgtc tttgtctgca cggtaagatc ctagcatgta gaactgatta 7080 actgccttgt aagggcagca gcccttctct acgggtagag agtatgcttg agcagctttt 7140 cgtagcgaag cgtgagtaag ggcaaaggtg tctctgacca tgactttgag aaattggtat 7200 ttgaagtcca tgtcgtcaca ggctccctgt tcccagagtt ggaagtctac ccgtttcttg 7260 taggcggggt tgggcaaagc gaaagtaaca tcattgaaga gaatcttacc ggctctgggc 7320 ataaaattgc gagtgatgcg gaaaggctgt ggtacttccg ctcgattgtt gatcacctgg 7380 gcagctagga cgatttcgtc gaaaccgttg atgttgtgtc ctacgatgta taattctatg 7440 aaacgcggcg tgcctctgac gtgaggtagc ttactgagct catcaaaggt taggtctgtg 7500 gggtcagata aggcgtagtg ttcgagagcc cattcgtgca ggtgaggatt tgcatgtagg 7560 aatgatgacc aaagatctac cgccagtgct gtttgtaact ggtcccgata ctgacgaaaa 7620 tgccggccaa ttgccatttt ttctggagtg acacagtaga aggttctggg gtcttgttgc 7680 catcgatccc acttgagttt aatggctaga tcgtgggcca tgttgacgag acgctcttct 7740 cctgagagtt tcatgaccag catgaaagga actagttgtt tgccaaagga tcccatccag 7800 gtgtaagttt ccacatcgta ggtcaggaag agtctttctg tgcgaggatg agagccgatc 7860 gggaagaact ggatttcctg ccaccagttg gaggattggc tgttgatgtg atggaagtag 7920 aagtttctgc ggcgcgccga gcattcgtgt ttgtgcttgt acagacggcc gcagtagtcg 7980 cagcgttgca cgggttgtat ctcgtgaatg agctgtacct ggcttccctt gacgagaaat 8040 ttcagtggga agccgaggcc tggcgattgt atctcgtgct cttctatatt cgctgtatcg 8100 gcctgttcat cttctgtttc gatggtggtc atgctgacga gcccccgcgg gaggcaagtc 8160 cagacctcgg cgcgggaggg gcggagctga aggacgagag cgcgcaggct ggagctgtcc 8220 agagtcctga gacgctgcgg actcaggtta gtaggtaggg acagaagatt aacttgcatg 8280 atcttttcca gggcgtgcgg gaggttcaga tggtacttga tttccacagg ttcgtttgta 8340 gagacgtcaa tggcttgcag ggttccgtgt cctttgggcg ccactaccgt acctttgttt 8400 tttcttttga tcggtggtgg ctctcttgct tcttgcatgc tcagaagcgg tgacggggac 8460 gcgcgccggg cggcagcggt tgttccggac ccgggggcat ggctggtagt ggcacgtcgg 8520 cgccgcgcac gggcaggttc tggtattgcg ctctgagaag acttgcgtgc gccaccacgc 8580 gtcgattgac gtcttgtatc tgacgtctct gggtgaaagc taccggcccc gtgagcttga 8640 acctgaaaga gagttcaaca gaatcaattt cggtatcgtt aacggcagct tgtctcagta 8700 tttcttgtac gtcaccagag ttgtcctggt aggcgatctc cgccatgaac tgctcgattt 8760 cttcctcctg aagatctccg cgacccgctc tttcgacggt ggccgcgagg tcattggaga 8820 tacggcccat gagttgggag aatgcattca tgcccgcctc gttccagacg cggctgtaaa 8880 ccacggcccc ctcggagtct cttgcgcgca tcaccacctg agcgaggtta agctccacgt 8940 gtctggtgaa gaccgcatag ttgcataggc gctgaaaaag gtagttgagt gtggtggcaa 9000 tgtgttcggc gacgaagaaa tacatgatcc atcgtctcag cggcatttcg ctaacatcgc 9060 ccagagcttc caagcgctcc atggcctcgt agaagtccac ggcaaaatta aaaaactggg 9120 agtttcgcgc ggacacggtc aattcctcct cgagaagacg gatgagttcg gctatggtgg 9180 cccgtacttc gcgttcgaag gctcccggga tctcttcttc ctcttctatc tcttcttcca 9240 ctaacatctc ttcttcgtct tcaggcgggg gcggaggggg cacgcggcga cgtcgacggc 9300 gcacgggcaa acggtcgatg aatcgttcaa tgacctctcc gcggcggcgg cgcatggttt 9360 cagtgacggc gcggccgttc tcgcgcggtc gcagagtaaa aacaccgccg cgcatctcct 9420 taaagtggtg actgggaggt tctccgtttg ggagggagag ggcgctgatt atacatttta 9480 ttaattggcc cgtagggact gcgcgcagag atctgatcgt gtcaagatcc acgggatctg 9540 aaaacctttc gacgaaagcg tctaaccagt cacagtcaca aggtaggctg agtacggctt 9600 cttgtgggcg ggggtggtta tgtgttcggt ctgggtcttc tgtttcttct tcatctcggg 9660 aaggtgagac gatgctgctg gtgatgaaat taaagtaggc agttctaaga cggcggatgg 9720 tggcgaggag caccaggtct ttgggtccgg cttgctggat acgcaggcga ttggccattc 9780 cccaagcatt atcctgacat ctagcaagat ctttgtagta gtcttgcatg agccgttcta 9840 cgggcacttc ttcctcaccc gttctgccat gcatacgtgt gagtccaaat ccgcgcattg 9900 gttgtaccag tgccaagtca gctacgactc tttcggcgag gatggcttgc tgtacttggg 9960 taagggtggc ttgaaagtca tcaaaatcca caaagcggtg gtaagcccct gtattaatgg 10020 tgtaagcaca gttggccatg actgaccagt taactgtctg gtgaccaggg cgcacgagct 10080 cggtgtattt aaggcgcgaa taggcgcggg tgtcaaagat gtaatcgttg caggtgcgca 10140 ccagatactg gtaccctata agaaaatgcg gcggtggttg gcggtagaga ggccatcgtt 10200 ctgtagctgg agcgccaggg gcgaggtctt ccaacataag gcggtgatag ccgtagatgt 10260 acctggacat ccaggtgatt cctgcggcgg tagtagaagc ccgaggaaac tcgcgtacgc 10320 ggttccaaat gttgcgtagc ggcatgaagt agttcattgt aggcacggtt tgaccagtga 10380 ggcgcgcgca gtcattgatg ctctatagac acggagaaaa tgaaagcgtt cagcgactcg 10440 actccgtagc ctggaggaac gtgaacgggt tgggtcgcgg tgtaccccgg ttcgagactt 10500 gtactcgagc cggccggagc cgcggctaac gtggtattgg cactcccgtc tcgacccagc 10560 ctacaaaaat ccaggatacg gaatcgagtc gttttgctgg tttccgaatg gcagggaagt 10620 gagtcctatt tttttttttt ttttgccgct cagatgcatc ccgtgctgcg acagatgcgc 10680 ccccaacaac agcccccctc gcagcagcag cagcagcagc aaccacaaaa ggctgtccct 10740 gcaactactg caactgccgc cgtgagcggt gcgggacagc ccgcctatga tctggacttg 10800 gaagagggcg aaggactggc acgtctaggt gcgccttcgc ccgagcggca tccgcgagtt 10860 caactgaaaa aagattctcg cgaggcgtat gtgccccaac agaacctatt tagagacaga 10920 agcggcgagg agccggagga gatgcgagct tcccgcttta acgcgggtcg tgagctgcgt 10980 cacggtttgg accgaagacg agtgttgcga gacgaggatt tcgaagttga tgaagtgaca 11040 gggatcagtc ctgccagggc acacgtggct gcagccaacc ttgtatcggc ttacgagcag 11100 acagtaaagg aagagcgtaa cttccaaaag tcttttaata atcatgtgcg aaccctgatt 11160 gcccgcgaag aagttaccct tggtttgatg catttgtggg atttgatgga agctatcatt 11220 cagaacccta ctagcaaacc tctgaccgcc cagctgtttc tggtggtgca acacagcaga 11280 gacaatgagg ctttcagaga ggcgctgctg aacatcaccg aacccgaggg gagatggttg 11340 tatgatctta tcaacattct acagagtatc atagtgcagg agcggagcct gggcctggcc 11400 gagaaggtag ctgccatcaa ttactcggtt ttgagcttgg gaaaatatta cgctcgcaaa 11460 atctacaaga ctccatacgt tcccatagac aaggaggtga agatagatgg gttctacatg 11520 cgcatgacgc tcaaggtctt gaccctgagc gatgatcttg gggtgtatcg caatgacaga 11580 atgcatcgcg cggttagcgc cagcaggagg cgcgagttaa gcgacaggga actgatgcac 11640 agtttgcaaa gagctctgac tggagctgga accgagggtg agaattactt cgacatggga 11700 gctgacttgc agtggcagcc tagtcgcagg gctctgagcg ccgcgacggc aggatgtgag 11760 cttccttaca tagaagaggc ggatgaaggc gaggaggaag agggcgagta cttggaagac 11820 tgatggcaca acccgtgttt tttgctagat ggaacagcaa gcaccggatc ccgcaatgcg 11880 ggcggcgctg cagagccagc cgtccggcat taactcctcg gacgattgga cccaggccat 11940 gcaacgtatc atggcgttga cgactcgcaa ccccgaagcc tttagacagc aaccccaggc 12000 caaccgtcta tcggccatca tggaagctgt agtgccttcc cgatctaatc ccactcatga 12060 gaaggtcctg gccatcgtga acgcgttggt ggagaacaaa gctattcgtc cagatgaggc 12120 cggactggta tacaacgctc tcttagaacg cgtggctcgc tacaacagta gcaatgtgca 12180 aaccaatttg gaccgtatga taacagatgt acgcgaagcc gtgtctcagc gcgaaaggtt 12240 ccagcgtgat gccaacctgg gttcgctggt ggcgttaaat gctttcttga gtactcagcc 12300 tgctaatgtg ccgcgtggtc aacaggatta tactaacttt ttaagtgctt tgagactgat 12360 ggtatcagaa gtacctcaga gcgaagtgta tcagtccggt cctgattact tctttcagac 12420 tagcagacag ggcttgcaga cggtaaatct gagccaagct tttaaaaacc ttaaaggttt 12480 gtggggagtg catgccccgg taggagaaag agcaaccgtg tctagcttgt taactccgaa 12540 ctcccgcctg ttattactgt tggtagctcc tttcaccgac agcggtagca tcgaccgtaa 12600 ttcctatttg ggttacctac taaacctgta tcgcgaagcc atagggcaaa gtcaggtgga 12660 cgagcagacc tatcaagaaa ttacccaagt cagtcgcgct ttgggacagg aagacactgg 12720 cagtttggaa gccactctga acttcttgct taccaatcgg tctcaaaaga tccctcctca 12780 atatgctctt actgcggagg aggagaggat ccttagatat gtgcagcaga gcgtgggatt 12840 gtttctgatg caagaggggg caactccgac tgcagcactg gacatgacag cgcgaaatat 12900 ggagcccagc atgtatgcca gtaaccgacc tttcattaac aaactgctgg actacttgca 12960 cagagctgcc gctatgaact ctgattattt caccaatgcc atcttaaacc cgcactggct 13020 gcccccacct ggtttctaca cgggcgaata tgacatgccc gaccctaatg acggatttct 13080 gtgggacgac gtggacagcg atgttttttc acctctttct gatcatcgca cgtggaaaaa 13140 ggaaggcggt gatagaatgc attcttctgc atcgctgtcc ggggtcatgg gtgctaccgc 13200 ggctgagccc gagtctgcaa gtccttttcc tagtctaccc ttttctctac acagtgtacg 13260 tagcagcgaa gtgggtagaa taagtcgccc gagtttaatg ggcgaagagg agtacctaaa 13320 cgattccttg ctcagaccgg caagagaaaa aaatttccca aacaatggaa tagaaagttt 13380 ggtggataaa atgagtagat ggaagactta tgctcaggat cacagagacg agcctgggat 13440 catggggact acaagtagag cgagccgtag acgccagcgc catgacagac agaggggtct 13500 tgtgtgggac gatgaggatt cggccgatga tagcagcgtg ttggacttgg gtgggagagg 13560 aaggggcaac ccgtttgctc atttgcgccc tcgcttgggt ggtatgttgt gaaaaaaaat 13620 aaaaaagaaa aactcaccaa ggccatggcg acgagcgtac gttcgttctt ctttattatc 13680 tgtgtctagt ataatgaggc gagtcgtgct aggcggagcg gtggtgtatc cggagggtcc 13740 tcctccttcg tacgagagcg tgatgcagca gcagcaggcg acggcggtga tgcaatcccc 13800 actggaggct ccctttgtgc ctccgcgata cctggcacct acggagggca gaaacagcat 13860 tcgttactcg gaactggcac ctcagtacga taccaccagg ttgtatctgg tggacaacaa 13920 gtcggcggac attgcttctc tgaactatca gaatgaccac agcaacttct tgaccacggt 13980 ggtgcagaac aatgacttta cccctacgga agccagcacc cagaccatta actttgatga 14040 acgatcgcgg tggggcggtc agctaaagac catcatgcat actaacatgc caaacgtgaa 14100 cgagtatatg tttagtaaca agttcaaagc gcgtgtgatg gtgtccagaa aacctcccga 14160 cggtgctgca gttggggata cttatgatca caagcaggat attttggaat atgagtggtt 14220 cgagtttact ttgccagaag gcaacttttc agttactatg actattgatt tgatgaacaa 14280 tgccatcata gataattact tgaaagtggg tagacagaat ggagtgcttg aaagtgacat 14340 tggtgttaag ttcgacacca ggaacttcaa gctgggatgg gatcccgaaa ccaagttgat 14400 catgcctgga gtgtatacgt atgaagcctt ccatcctgac attgtcttac tgcctggctg 14460 cggagtggat tttaccgaga gtcgtttgag caaccttctt ggtatcagaa aaaaacagcc 14520 atttcaagag ggttttaaga ttttgtatga agatttagaa ggtggtaata ttccggccct 14580 cttggatgta gatgcctatg agaacagtaa gaaagaacaa aaagccaaaa tagaagctgc 14640 tacagctgct gcagaagcta aggcaaacat agttgccagc gactctacaa gggttgctaa 14700 cgctggagag gtcagaggag acaattttgc gccaacacct gttccgactg cagaatcatt 14760 attggccgat gtgtctgatg gaacggacgt gaaactcact attcaacctg tagaaaaaga 14820 tagtaagaat agaagctata atgtgttgga agacaaaatc aacacagcct atcgcagttg 14880 gtatctttcg tacaattatg gcgatcccga aaaaggagtg cgttcctgga cattgctcac 14940 cacctcagat gtcacctgcg gagcagagca ggtttactgg tcgcttccag acatgatgaa 15000 ggatcctgtc actttccgct ccactagaca agtcagtaac taccctgtgg tgggtgcaga 15060 gcttatgccc gtcttctcaa agagcttcta caacgaacaa gctgtgtact cccagcagct 15120 ccgccagtcc acctcgctta cgcacgtctt caaccgcttt cctgagaacc agattttaat 15180 ccgtccgccg gcgcccacca ttaccaccgt cagtgaaaac gttcctgctc tcacagatca 15240 cgggaccctg ccgttgcgca gcagtatccg gggagtccaa cgtgtgaccg ttactgacgc 15300 cagacgccgc acctgtccct acgtgtacaa ggcactgggc atagtcgcac cgcgcgtcct 15360 ttcaagccgc actttctaaa aaaaaaatgt ccattcttat ctcgcccagt aataacaccg 15420 gttggggtct gcgcgctcca agcaagatgt acggaggcgc acgcaaacgt tctacccaac 15480 atcccgtgcg tgttcgcgga cattttcgcg ctccatgggg tgccctcaag ggccgcactc 15540 gcgttcgaac caccgtcgat gatgtaatcg atcaggtggt tgccgacgcc cgtaattata 15600 ctcctactgc gcctacatct actgtggatg cagttattga cagtgtagtg gctgacgctc 15660 gcaactatgc tcgacgtaag agccggcgaa ggcgcattgc cagacgccac cgagctacca 15720 ctgccatgcg agccgcaaga gctctgctac gaagagctag acgcgtgggg cgaagagcca 15780 tgcttagggc ggccagacgt gcagcttcgg gcgccagcgc cggcaggtcc cgcaggcaag 15840 cagccgctgt cgcagcggcg actattgccg acatggccca atcgcgaaga ggcaatgtat 15900 actgggtgcg tgacgctgcc accggtcaac gtgtacccgt gcgcacccgt ccccctcgca 15960 cttagaagat actgagcagt ctccgatgtt gtgtcccagc ggcgaggatg tccaagcgca 16020 aatacaagga agaaatgctg caggttatcg cacctgaagt ctacggccaa ccgttgaagg 16080 atgaaaaaaa accccgcaaa atcaagcggg ttaaaaagga caaaaaagaa gaggaagatg 16140 gcgatgatgg gctggcggag tttgtgcgcg agtttgcccc acggcgacgc gtgcaatggc 16200 gtgggcgcaa agttcgacat gtgttgagac ctggaacttc ggtggtcttt acacccggcg 16260 agcgttcaag cgctactttt aagcgttcct atgatgaggt gtacggggat gatgatattc 16320 ttgagcaggc ggctgaccga ttaggcgagt ttgcttatgg caagcgtagt agaataactt 16380 ccaaggatga gacagtgtca atacccttgg atcatggaaa tcccacccct agtcttaaac 16440 cggtcacttt gcagcaagtg ttacccgtaa ctccgcgaac aggtgttaaa cgcgaaggtg 16500 aagatttgta tcccactatg caactgatgg tacccaaacg ccagaagttg gaggacgttt 16560 tggagaaagt aaaagtggat ccagatattc aacctgaggt taaagtgaga cccattaagc 16620 aggtagcgcc tggtctgggg gtacaaactg tagacattaa gattcccact gaaagtatgg 16680 aagtgcaaac tgaacccgca aagcctactg ccacctccac tgaagtgcaa acggatccat 16740 ggatgcccat gcctattaca actgacgccg ccggtcccac tcgaagatcc cgacgaaagt 16800 acggtccagc aagtctgttg atgcccaatt atgttgtaca cccatctatt attcctactc 16860 ctggttaccg aggcactcgc tactatcgca gccgaaacag tacctcccgc cgtcgccgca 16920 agacacctgc aaatcgcagt cgtcgccgta gacgcacaag caaaccgact cccggcgccc 16980 tggtgcggca agtgtaccgc aatggtagtg cggaaccttt gacactgccg cgtgcgcgtt 17040 accatccgag tatcatcact taatcaatgt tgccgctgcc tccttgcaga tatggccctc 17100 acttgtcgcc ttcgcgttcc catcactggt taccgaggaa gaaactcgcg ccgtagaaga 17160 gggatgttgg gacgcggaat gcgacgctac aggcgacggc gtgctatccg caagcaattg 17220 cggggtggtt ttttaccagc cttaattcca attatcgctg ctgcaattgg cgcgatacca 17280 ggcatagctt ccgtggcggt tcaggcctcg caacgacatt gacattggaa aaaaaacgta 17340 taaataaaaa aaaatacaat ggactctgac actcctggtc ctgtgactat gttttcttag 17400 agatggaaga catcaatttt tcatccttgg ctccgcgaca cggcacgaag ccgtacatgg 17460 gcacctggag cgacatcggc acgagccaac tgaacggggg cgccttcaat tggagcagta 17520 tctggagcgg gcttaaaaat tttggctcaa ccataaaaac atacgggaac aaagcttgga 17580 acagcagtac aggacaggcg cttagaaata aacttaaaga ccagaacttc caacaaaaag 17640 tagtcgatgg gatagcttcc ggcatcaatg gagtggtaga tttggctaac caggctgtgc 17700 agaaaaagat aaacagtcgt ttggacccgc cgccagcaac cccaggtgaa atgcaagtgg 17760 aggaagaaat tcctccgcca gaaaaacgag gcgacaagcg tccgcgtccc gatttggaag 17820 agacgctggt gacgcgcgta gatgaaccgc cttcttatga ggaagcaacg aagcttggaa 17880 tgcccaccac tagaccgata gccccaatgg ccaccggggt gatgaaacct tctcagttgc 17940 atcgacccgt caccttggat ttgccccctc cccctgctgc tactgctgta cccgcttcta 18000 agcctgtcgc tgccccgaaa ccagtcgccg tagccaggtc acgtcccggg ggcgctcctc 18060 gtccaaatgc gcactggcaa aatactctga acagcatcgt gggtctaggc gtgcaaagtg 18120 taaaacgccg tcgctgcttt taattaaata tggagtagcg cttaacttgc ctatctgtgt 18180 atatgtgtca ttacacgccg tcacagcagc agaggaaaaa aggaagaggt cgtgcgtcga 18240 cgctgagtta ctttcaagat ggccacccca tcgatgctgc cccaatgggc atacatgcac 18300 atcgccggac aggatgcttc ggagtacctg agtccgggtc tggtgcagtt cgcccgcgcc 18360 acagacacct acttcaatct gggaaataag tttagaaatc ccaccgtagc gccgacccac 18420 gatgtgacca ccgaccgtag ccagcggctc atgttgcgct tcgtgcccgt tgaccgggag 18480 gacaatacat actcttacaa agtgcggtac accctggccg tgggcgacaa cagagtgctg 18540 gatatggcca gcacgttctt tgacattagg ggcgtgttgg acagaggtcc cagtttcaaa 18600 ccctattctg gtacggctta caactctctg gctcctaaag gcgctccaaa tgcatctcaa 18660 tggattgcaa aaggcgtacc aactgcagca gccgcaggca atggtgaaga agaacatgaa 18720 acagaggaga aaactgctac ttacactttt gccaatgctc ctgtaaaagc cgaggctcaa 18780 attacaaaag agggcttacc aataggtttg gagatttcag ctgaaaacga atctaaaccc 18840 atctatgcag ataaacttta tcagccagaa cctcaagtgg gagatgaaac ttggactgac 18900 ctagacggaa aaaccgaaga gtatggaggc agggctctaa agcctactac taacatgaaa 18960 ccctgttacg ggtcctatgc gaagcctact aatttaaaag gtggtcaggc aaaaccgaaa 19020 aactcggaac cgtcgagtga aaaaattgaa tatgatattg acatggaatt ttttgataac 19080 tcatcgcaaa gaacaaactt cagtcctaaa attgtcatgt atgcagaaaa tgtaggtttg 19140 gaaacgccag acactcatgt agtgtacaaa cctggaacag aagacacaag ttccgaagct 19200 aatttgggac aacagtctat gcccaacaga cccaactaca ttggcttcag agataacttt 19260 attggactca tgtactataa cagtactggt aacatggggg tgctggctgg tcaagcgtct 19320 cagttaaatg cagtggttga cttgcaggac agaaacacag aactttctta ccaactcttg 19380 cttgactctc tgggcgacag aaccagatac tttagcatgt ggaatcaggc tgtggacagt 19440 tatgatcctg atgtacgtgt tattgaaaat catggtgtgg aagatgaact tcccaactat 19500 tgttttccac tggacggcat aggtgttcca acaaccagtt acaaatcaat agttccaaat 19560 ggagaagata ataataattg gaaagaacct gaagtaaatg gaacaagtga gatcggacag 19620 ggtaatttgt ttgccatgga aattaacctt caagccaatc tatggcgaag tttcctttat 19680 tccaatgtgg ctctgtatct cccagactcg tacaaataca ccccgtccaa tgtcactctt 19740 ccagaaaaca aaaacaccta cgactacatg aacgggcggg tggtgccgcc atctctagta 19800 gacacctatg tgaacattgg tgccaggtgg tctctggatg ccatggacaa tgtcaaccca 19860 ttcaaccacc accgtaacgc tggcttgcgt taccgatcta tgcttctggg taacggacgt 19920 tatgtgcctt tccacataca agtgcctcaa aaattcttcg ctgttaaaaa cctgctgctt 19980 ctcccaggct cctacactta tgagtggaac tttaggaagg atgtgaacat ggttctacag 20040 agttccctcg gtaacgacct gcgggtagat ggcgccagca tcagtttcac gagcatcaac 20100 ctctatgcta cttttttccc catggctcac aacaccgctt ccacccttga agccatgctg 20160 cggaatgaca ccaatgatca gtcattcaac gactacctat ctgcagctaa catgctctac 20220 cccattcctg ccaatgcaac caatattccc atttccattc cttctcgcaa ctgggcggct 20280 ttcagaggct ggtcatttac cagactgaaa accaaagaaa ctccctcttt ggggtctgga 20340 tttgacccct actttgtcta ttctggttct attccctacc tggatggtac cttctacctg 20400 aaccacactt ttaagaaggt ttccatcatg tttgactctt cagtgagctg gcctggaaat 20460 gacaggttac tatctcctaa cgaatttgaa ataaagcgca ctgtggatgg cgaaggctac 20520 aacgtagccc aatgcaacat gaccaaagac tggttcttgg tacagatgct cgccaactac 20580 aacatcggct atcagggctt ctacattcca gaaggataca aagatcgcat gtattcattt 20640 ttcagaaact tccagcccat gagcaggcag gtggttgatg aggtcaatta caaagacttc 20700 aaggccgtcg ccatacccta ccaacacaac aactctggct ttgtgggtta catggctccg 20760 accatgcgcc aaggtcaacc ctatcccgct aactatccct atccactcat tggaacaact 20820 gccgtaaata gtgttacgca gaaaaagttc ttgtgtgaca gaaccatgtg gcgcataccg 20880 ttctcgagca acttcatgtc tatgggggcc cttacagact tgggacagaa tatgctctat 20940 gccaactcag ctcatgctct ggacatgacc tttgaggtgg atcccatgga tgagcccacc 21000 ctgctttatc ttctcttcga agttttcgac gtggtcagag tgcatcagcc acaccgcggc 21060 atcatcgagg cagtctacct gcgtacaccg ttctcggccg gtaacgctac cacgtaagaa 21120 gcttcttgct tcttgcaaat agcagctgca accatggcct gcggatccca aaacggctcc 21180 agcgagcaag agctcagagc cattgtccaa gacctgggtt gcggacccta ttttttggga 21240 acctacgata agcgcttccc ggggttcatg gcccccgata agctcgcctg tgccattgta 21300 aatacggccg gacgtgagac ggggggagag cactggttgg ctttcggttg gaacccacgt 21360 tctaacacct gctacctttt tgatcctttt ggattctcgg atgatcgtct caaacagatt 21420 taccagtttg aatatgaggg tctcctgcgc cgcagcgctc ttgctaccaa ggaccgctgt 21480 attacgctgg aaaaatctac ccagaccgtg cagggccccc gttctgccgc ctgcggactt 21540 ttctgctgca tgttccttca cgcctttgtg cactggcctg accgtcccat ggacggaaac 21600 cccaccatga aattgctaac tggagtgcca aacaacatgc ttcattctcc taaagtccag 21660 cccaccctgt gtgacaatca aaaagcactc taccattttc ttaataccca ttcgccttat 21720 tttcgctctc atcgtacaca catcgaaagg gccactgcgt tcgaccgtat ggatgttcaa 21780 taatgactca tgtaaacaac gtgttcaata aacatcactt tattttttta catgtatcaa 21840 ggctctggat tacttattta tttacaagtc gaatgggttc tgacgagaat cagaatgacc 21900 cgcaggcagt gatacgttgc ggaactgata cttgggttgc cacttgaatt cgggaatcac 21960 caacttggga accggtatat cgggcaggat gtcactccac agctttctgg tcagctgcaa 22020 agctccaagc aggtcaggag ccgaaatctt gaaatcacaa ttaggaccag tgctctgagc 22080 gcgagagttg cggtacaccg gattgcagca ctgaaacacc atcagcgacg gatgtctcac 22140 gcttgccagc acggtgggat ctgcaatcat gcccacatcc agatcttcag cattggcaat 22200 gctgaacggg gtcatcttgc aggtctgcct acccatggcg ggcacccaat taggcttgtg 22260 gttgcaatcg cagtgcaggg ggatcagtat catcttggcc tgatcctgtc tgattcctgg 22320 atacacggct ctcatgaaag catcatattg cttgaaagcc tgctgggctt tactaccctc 22380 ggtataaaac atcccgcagg acctgctcga aaactggtta gctgcacagc cggcatcatt 22440 cacacagcag cgggcgtcat tgttggctat ttgcaccaca cttctgcccc agcggttttg 22500 ggtgattttg gttcgctcgg gattctcctt taaggctcgt tgtccgttct cgctggccac 22560 atccatctcg ataatctgct ccttctgaat cataatattg ccatgcaggc acttcagctt 22620 gccctcataa tcattgcagc catgaggcca caacgcacag cctgtacatt cccaattatg 22680 gtgggcgatc tgagaaaaag aatgtatcat tccctgcaga aatcttccca tcatcgtgct 22740 cagtgtcttg tgactagtga aagttaactg gatgcctcgg tgctcttcgt ttacgtactg 22800 gtgacagatg cgcttgtatt gttcgtgttg ctcaggcatt agtttaaaac aggttctaag 22860 ttcgttatcc agcctgtact tctccatcag cagacacatc acttccatgc ctttctccca 22920 agcagacacc aggggcaagc taatcggatt cttaacagtg caggcagcag ctcctttagc 22980 cagagggtca tctttagcga tcttctcaat gcttcttttg ccatccttct caacgatgcg 23040 cacgggcggg tagctgaaac ccactgctac aagttgcgcc tcttctcttt cttcttcgct 23100 gtcttgactg atgtcttgca tggggatatg tttggtcttc cttggcttct ttttgggggg 23160 tatcggagga ggaggactgt cgctccgttc cggagacagg gaggattgtg acgtttcgct 23220 caccattacc aactgactgt cggtagaaga acctgacccc acacggcgac aggtgttttt 23280 cttcgggggc agaggtggag gcgattgcga agggctgcgg tccgacctgg aaggcggatg 23340 actggcagaa ccccttccgc gttcgggggt gtgctccctg tggcggtcgc ttaactgatt 23400 tccttcgcgg ctggccattg tgttctccta ggcagagaaa caacagacat ggaaactcag 23460 ccattgctgt caacatcgcc acgagtgcca tcacatctcg tcctcagcga cgaggaaaag 23520 gagcagagct taagcattcc accgcccagt cctgccacca cctctaccct agaagataag 23580 gaggtcgacg catctcatga catgcagaat aaaaaagcga aagagtctga gacagacatc 23640 gagcaagacc cgggctatgt gacaccggtg gaacacgagg aagagttgaa acgctttcta 23700 gagagagagg atgaaaactg cccaaaacag cgagcagata actatcacca agatgctgga 23760 aatagggatc agaacaccga ctacctcata gggcttgacg gggaagacgc gctccttaaa 23820 catctagcaa gacagtcgct catagtcaag gatgcattat tggacagaac tgaagtgccc 23880 atcagtgtgg aagagctcag ctgcgcctac gagcttaacc ttttttcacc tcgtactccc 23940 cccaaacgtc agccaaacgg cacctgcgag ccaaatcctc gcttaaactt ttatccagct 24000 tttgctgtgc cagaagtact ggctacctat cacatctttt ttaaaaatca aaaaattcca 24060 gtctcctgcc gcgctaatcg cacccgcgcc gatgccctac tcaatctggg acctggttca 24120 cgcttacctg atatagcttc cttggaagag gttccaaaga tcttcgaggg tctgggcaat 24180 aatgagactc gggccgcaaa tgctctgcaa aagggagaaa atggcatgga tgagcatcac 24240 agcgttctgg tggaattgga aggcgataat gccagactcg cagtactcaa gcgaagcgtc 24300 gaggtcacac acttcgcata tcccgctgtc aacctgcccc ctaaagtcat gacggcggtc 24360 atggaccagt tactcattaa gcgcgcaagt cccctttcag aagacatgca tgacccagat 24420 gcctgtgatg agggtaaacc agtggtcagt gatgagcagc taacccgatg gctgggcacc 24480 gactctcccc gggatttgga agagcgtcgc aagcttatga tggccgtggt gctggttacc 24540 gtagaactag agtgtctccg acgtttcttt accgattcag aaaccttgcg caaactcgaa 24600 gagaatctgc actacacttt tagacacggc tttgtgcggc aggcatgcaa gatatctaac 24660 gtggaactca ccaacctggt ttcctacatg ggtattctgc atgagaatcg cctaggacaa 24720 agcgtgctgc acagcaccct taagggggaa gcccgccgtg attacatccg cgattgtgtc 24780 tatctctacc tgtgccacac gtggcaaacc ggcatgggtg tatggcagca atgtttagaa 24840 gaacagaact tgaaagagct tgacaagctc ttacagaaat ctcttaaggt tctgtggaca 24900 gggttcgacg agcgcaccgt cgcttccgac ctggcagacc tcatcttccc agagcgtctc 24960 agggttactt tgcgaaacgg attgcctgac tttatgagcc agagcatgct taacaatttt 25020 cgctctttca tcctggaacg ctccggtatc ctgcccgcca cctgctgcgc actgccctcc 25080 gactttgtgc ctctcaccta ccgcgagtgc cccccgccgc tatggagtca ctgctacctg 25140 ttccgtctgg ccaactatct ctcctaccac tcggatgtga tcgaggatgt gagcggagac 25200 ggcttgctgg agtgccactg ccgctgcaat ctgtgcacgc cccaccggtc cctagcttgc 25260 aacccccagt tgatgagcga aacccagata ataggcacct ttgaattgca aggccccagc 25320 agccaaggcg atgggtcttc tcctgggcaa agtttaaaac tgaccccggg actgtggacc 25380 tccgcctact tgcgcaagtt tgctccggaa gattaccacc cctatgaaat caagttctat 25440 gaggaccaat cacagcctcc aaaggccgaa ctttcggctt gcgtcatcac ccagggggca 25500 attctggccc aattgcaagc catccaaaaa tcccgccaag aatttctact gaaaaagggt 25560 aagggggtct accttgaccc ccagaccggc gaggaactca acacaaggtt ccctcaggat 25620 gtcccaacga cgagaaaaca agaagttgaa ggtgcagccg ccgcccccag aagatatgga 25680 ggaagattgg gacagtcagg cagaggaggc ggaggaggac agtctggagg acagtctgga 25740 ggaagacagt ttggaggagg aaaacgagga ggcagaggag gtggaagaag taaccgccga 25800 caaacagtta tcctcggctg cggagacaag caacagcgct accatctccg ctccgagtcg 25860 aggaacccgg cggcgtccca gcagtagatg ggacgagacc ggacgcttcc cgaacccaac 25920 cagcgcttcc aagaccggta agaaggatcg gcagggatac aagtcctggc gggggcataa 25980 gaatgccatc atctcctgct tgcatgagtg cgggggcaac atatccttca cgcggcgcta 26040 cttgctattc caccatgggg tgaactttcc gcgcaatgtt ttgcattact accgtcacct 26100 ccacagcccc tactatagcc agcaaatccc gacagtctcg acagataaag acagcggcgg 26160 cgacctccaa cagaaaacca gcagcggcag ttagaaaata cacaacaagt gcagcaacag 26220 gaggattaaa gattacagcc aacgagccag cgcaaacccg agagttaaga aatcggatct 26280 ttccaaccct gtatgccatc ttccagcaga gtcggggtca agagcaggaa ctgaaaataa 26340 aaaaccgatc tctgcgttcg ctcaccagaa gttgtttgta tcacaagagc gaagatcaac 26400 ttcagcgcac tctcgaggac gccgaggctc tcttcaacaa gtactgcgcg ctgactctta 26460 aagagtaggc agcgaccgcg cttattcaaa aaaggcggga attacatcat cctcgacatg 26520 agtaaagaaa ttcccacgcc ttacatgtgg agttatcaac cccaaatggg attggcagca 26580 ggcgcctccc aggactactc cacccgcatg aattggctca gcgccgggcc ttctatgatt 26640 tctcgagtta atgatatacg cgcctaccga aaccaaatac ttttggaaca gtcagctctt 26700 accaccacgc cccgccaaca ccttaatccc agaaattggc ccgccgccct agtgtaccag 26760 gaaagtcccg ctcccaccac tgtattactt cctcgagacg cccaggccga agtccaaatg 26820 actaatgcag gtgcgcagtt agctggcggc tccaccctat gtcgtcacag gcctcggcat 26880 aatataaaac gcctgatgat cagaggccga ggtatccagc tcaacgacga gtcggtgagc 26940 tctccgcttg gtctacgacc agacggaatc tttcagattg ccggctgcgg gagatcttcc 27000 ttcacccctc gtcaggctgt tctgactttg gaaagttcgt cttcgcaacc ccgctcgggc 27060 ggaatcggga ccgttcaatt tgtagaggag tttactccct ctgtctactt caaccccttc 27120 tccggatctc ctgggcacta cccggacgag ttcataccga acttcgacgc gattagcgag 27180 tcagtggacg gctacgattg atgtctggtg acgcggctga gctatctcgg ctgcgacatc 27240 tagaccactg ccgccgcttt cgctgctttg cccgggaact tattgagttc atctacttcg 27300 aactccccaa ggatcaccct caaggtccgg cccacggagt gcggattact atcgaaggca 27360 aaatagactc tcgcctgcaa cgaattttct cccagcggcc cgtgctgatc gagcgagacc 27420 agggaaacac cacggtttcc atctactgca tttgtaatca ccccggattg catgaaagcc 27480 tttgctgtct tatgtgtact gagtttaata aaaactgaat taagactctc ctacggactg 27540 ccgcttcttc aacccggatt ttacaaccag aagaacaaaa cttttcctgt cgtccaggac 27600 tctgttaact tcacctttcc tactcacaaa ctagaagctc aacgactaca ccgcttttcc 27660 agaagcattt tccctactaa tactactttc aaaaccggag gtgagctcca cggtctccct 27720 acagaaaacc cttgggtgga agcgggcctt gtagtactag gaattcttgc gggtgggctt 27780 gtgattattc tttgctacct atacacacct tgcttcactt tcctagtggt gttgtggtat 27840 tggtttaaaa aatggggccc atactagtct tgcttgtttt actttcgctt ttggaaccgg 27900 gttctgccaa ttacgatcca tgtctagact ttgacccaga aaactgcaca cttacttttg 27960 cacccgacac aagccgcatc tgtggagttc ttattaagtg cggatgggaa tgcaggtccg 28020 ttgaaattac acacaataac aaaacctgga acaatacctt atccaccaca tgggagccag 28080 gagttcccga gtggtacact gtctctgtcc gaggtcctga cggttccatc cgcattagta 28140 acaacacttt cattttttct gaaatgtgcg atctggccat gttcatgagc aaacagtatt 28200 ctctatggcc tcctagcaag gacaacatcg taacgttctc cattgcttat tgcttgtgcg 28260 cttgccttct tactgcttta ctgtgcgtat gcatacacct gcttgtaacc actcgcatca 28320 aaaacgccaa taacaaagaa aaaatgcctt aacctctttc tgtttacaga catggcttct 28380 cttacatctc tcatatttgt cagcattgtc actgccgctc acggacaaac agtcgtctct 28440 atcccactag gacataatta cactctcata ggacccccaa tcacttcaga ggtcatctgg 28500 accaaactgg gaagcgttga ttactttgat ataatctgta acaaaacaaa accaataata 28560 gtaacttgca acatacaaaa tcttacattg attaatgtta gcaaagttta cagcggttac 28620 tattatggtt atgacagata cagtagtcaa tatagaaatt acttggttcg tgttacccag 28680 ttgaaaacca cgaaaatgcc aaatatggca aagattcgat ccgatgacaa ttctctagaa 28740 acttttacat ctcccaccac acccgacgaa aaaaacatcc cagattcaat gattgcaatt 28800 gttgcagcgg tggcagtggt gatggcacta ataataatat gcatgctttt atatgcttgt 28860 cgctacaaaa agtttcatcc taaaaaacaa gatctcctac taaggcttaa catttaattt 28920 ctttttatac agccatggtt tccactacca cattccttat gcttactagt ctcgcaactc 28980 tgacttctgc tcgctcacac ctcactgtaa ctataggctc aaactgcaca ctaaaaggac 29040 ctcaaggtgg tcatgtcttt tggtggagaa tatatgacaa tggatggttt acaaaaccat 29100 gtgaccaacc tggtagattt ttctgcaacg gcagagacct aaccattatc aacgtgacag 29160 caaatgacaa aggcttctat tatggaaccg actataaaag tagtttagat tataacatta 29220 ttgtactgcc atctaccact ccagcacccc gcacaactac tttctctagc agcagtgtcg 29280 ctaacaatac aatttccaat ccaacctttg ccgcgctttt aaaacgcact gtgaataatt 29340 ctacaacttc acatacaaca atttccactt caacaatcag catcatcgct gcagtgacaa 29400 ttggaatatc tattcttgtt tttaccataa cctactacgc ctgctgctat agaaaagaca 29460 aacataaagg tgatccatta cttagatttg atatttaatt tgttcttttt ttttatttac 29520 agtatggtga acaccaatca tggtacctag aaatttcttc ttcaccatac tcatctgtgc 29580 ttttaatgtt tgcgctactt tcacagcagt agccacagca accccagact gtataggagc 29640 atttgcttcc tatgcacttt ttgcttttgt tacttgcatc tgcgtatgta gcatagtctg 29700 cctggttatt aattttttcc aacttctaga ctggatcctt gtgcgaattg cctacctgcg 29760 ccaccatccc gaataccgca accaaaatat cgcggcactt cttagactca tctaaaacca 29820 tgcaggctat actaccaata tttttgcttc tattgcttcc ctacgctgtc tcaaccccag 29880 ctgcctatag tactccacca gaacacctta gaaaatgcaa attccaacaa ccgtggtcat 29940 ttcttgcttg ctatcgagaa aaatcagaaa tccccccaaa tttaataatg attgctggaa 30000 taattaatat aatctgttgc accataattt catttttgat atacccccta tttgattttg 30060 gctggaatgc tcccaatgca catgatcatc cacaagaccc agaggaacac attcccccac 30120 aaaacatgca acatccaata gcgctaatag attacgaaag tgaaccacaa cccccactac 30180 tccctgctat tagttacttc aacctaaccg gcggagatga ctgaaacact caccacctcc 30240 aattccgccg aggatctgct cgatatggac ggccgcgtct cagaacaacg acttgcccaa 30300 ctacgcatcc gccagcagca ggaacgcgtg gccaaagagc tcagagatgt catccaaatt 30360 caccaatgca aaaaaggcat attctgtttg gtaaaacaag ccaagatatc ctacgagatc 30420 accgctactg accatcgcct ctcttacgaa cttggccccc aacgacaaaa atttacctgc 30480 atggtgggaa tcaaccccat agttatcacc caacaaagtg gagatactaa gggttgcatt 30540 cactgctcct gcgattccat cgagtgcacc tacaccctgc tgaagaccct atgcggccta 30600 agagacctgc taccaatgaa ttaaaaaaaa atgattaata aaaaatcact tacttgaaat 30660 cagcaataag gtctctgttg aaattttctc ccagcagcac ctcacttccc tcttcccaac 30720 tctggtattc taaaccccgt tcagcggcat actttctcca tactttaaag gggatgtcaa 30780 attttagctc ctctcctgta cccacaatct tcatgtcttt cttcccagat gaccaagaga 30840 gtccggctca gtgactcctt caaccctgtc tacccctatg aagatgaaag cacctcccaa 30900 caccccttta taaacccagg gtttatttcc ccaaatggct tcacacaaag cccagacgga 30960 gttcttactt taaaatgttt aaccccacta acaaccacag gcggatctct acagctaaaa 31020 gtgggagggg gacttacagt ggatgacact gatggtacct tacaagaaaa catacgtgct 31080 acagcaccca ttactaaaaa taatcactct gtagaactat ccattggaaa tggattagaa 31140 actcaaaaca ataaactatg tgccaaattg ggaaatgggt taaaatttaa caacggtgac 31200 atttgtataa aggatagtat taacacctta tggactggaa taaaccctcc acctaactgt 31260 caaattgtgg aaaacactaa tacaaatgat ggcaaactta ctttagtatt agtaaaaaat 31320 ggagggcttg ttaatggcta cgtgtctcta gttggtgtat cagacactgt gaaccaaatg 31380 ttcacacaaa agacagcaaa catccaatta agattatatt ttgactcttc tggaaatcta 31440 ttaactgagg aatcagactt aaaaattcca cttaaaaata aatcttctac agcgaccagt 31500 gaaactgtag ccagcagcaa agcctttatg ccaagtacta cagcttatcc cttcaacacc 31560 actactaggg atagtgaaaa ctacattcat ggaatatgtt actacatgac tagttatgat 31620 agaagtctat ttcccttgaa catttctata atgctaaaca gccgtatgat ttcttccaat 31680 gttgcctatg ccatacaatt tgaatggaat ctaaatgcaa gtgaatctcc agaaagcaac 31740 atagctacgc tgaccacatc cccctttttc ttttcttaca ttacagaaga cgacaactaa 31800 aataaagttt aagtgttttt atttaaaatc acaaaattcg agtagttatt ttgcctccac 31860 cttcccattt gacagaatac accaatctct ccccacgcac agctttaaac atttggatac 31920 cattagagat agacattgtt ttagattcca cattccaaac agtttcagag cgagccaatc 31980 tggggtcagt gatagataaa aatccatcgc gatagtcttt taaagcgctt tcacagtcca 32040 actgctgcgg atgcgactcc ggagtttgga tcacggtcat ctggaagaag aacgatggga 32100 atcataatcc gaaaacggta tcggacgatt gtgtctcatc aaacccacaa gcagccgctg 32160 tctgcgtcgc tccgtgcgac tgctgtttat gggatcaggg tccacagttt cctgaagcat 32220 gattttaata gcccttaaca tcaactttct ggtgcgatgc gcgcagcaac gcattctgat 32280 ttcactcaaa tctttgcagt aggtacaaca cattattaca atattgttta ataaaccata 32340 attaaaagcg ctccagccaa aactcatatc tgatataatc gcccctgcat gaccatcata 32400 ccaaagttta atataaatta aatgacgttc cctcaaaaac acactaccca catacatgat 32460 ctcttttggc atgtgcatat taacaatctg tctgtaccat ggacaacgtt ggttaatcat 32520 gcaacccaat ataaccttcc ggaaccacac tgccaacacc gctcccccag ccatgcattg 32580 aagtgaaccc tgctgattac aatgacaatg aagaacccaa ttctctcgac cgtgaatcac 32640 ttgagaatga aaaatatcta tagtggcaca acatagacat aaatgcatgc atcttctcat 32700 aatttttaac tcctcaggat ttagaaacat atcccaggga ataggaagct cttgcagaac 32760 agtaaagctg gcagaacaag gaagaccacg aacacaactt acactatgca tagtcatagt 32820 atcacaatct ggcaacagcg ggtggtcttc agtcatagaa gctcgggttt cattttcctc 32880 acaacgtggt aactgggctc tggtgtaagg gtgatgtctg gcgcatgatg tcgagcgtgc 32940 gcgcaacctt gtcataatgg agttgcttcc tgacattctc gtattttgta tagcaaaacg 33000 cggccctggc agaacacact cttcttcgcc ttctatcctg ccgcttagcg tgttccgtgt 33060 gatagttcaa gtacagccac actcttaagt tggtcaaaag aatgctggct tcagttgtaa 33120 tcaaaactcc atcgcatcta attgttctga ggaaatcatc cacggtagca tatgcaaatc 33180 ccaaccaagc aatgcaactg gattgcgttt caagcaggag aggagaggga agagacggaa 33240 gaaccatgtt aatttttatt ccaaacgatc tcgcagtact tcaaattgta gatcgcgcag 33300 atggcatctc tcgcccccac tgtgttggtg aaaaagcaca gctaaatcaa aagaaatgcg 33360 attttcaagg tgctcaacgg tggcttccaa caaagcctcc acgcgcacat ccaagaacaa 33420 aagaatacca aaagaaggag cattttctaa ctcctcaatc atcatattac attcctgcac 33480 cattcccaga taattttcag ctttccagcc ttgaattatt cgtgtcagtt cttgtggtaa 33540 atccaatcca cacattacaa acaggtcccg gagggcgccc tccaccacca ttcttaaaca 33600 caccctcata atgacaaaat atcttgctcc tgtgtcacct gtagcgaatt gagaatggca 33660 acatcaattg acatgccctt ggctctaagt tcttctttaa gttctagttg taaaaactct 33720 ctcatattat caccaaactg cttagccaga agccccccgg gaacaagagc aggggacgct 33780 acagtgcagt acaagcgcag acctccccaa ttggctccag caaaaacaag attggaataa 33840 gcatattggg aaccaccagt aatatcatcg aagttgctgg aaatataatc aggcagagtt 33900 tcttgtagaa attgaataaa agaaaaattt gccaaaaaaa cattcaaaac ctctgggatg 33960 caaatgcaat aggttaccgc gctgcgctcc aacattgtta gttttgaatt agtctgcaaa 34020 aataaaaaaa aaacaagcgt catatcatag tagcctgacg aacaggtgga taaatcagtc 34080 tttccatcac aagacaagcc acagggtctc cagctcgacc ctcgtaaaac ctgtcatcgt 34140 gattaaacaa cagcaccgaa agttcctcgc ggtgaccagc atgaataagt cttgatgaag 34200 catacaatcc agacatgtta gcatcagtta aggagaaaaa acagccaaca tagcctttgg 34260 gtataattat gcttaatcgt aagtatagca aagccacccc tcgcggatac aaagtaaaag 34320 gcacaggaga ataaaaaata taattatttc tctgctgctg tttaggcaac gtcgcccccg 34380 gtccctctaa atacacatac aaagcctcat cagccatggc ttaccagaga aagtacagcg 34440 ggcacacaaa ccacaagctc taaagtcact ctccaacctc tccacaatat atatacacaa 34500 gccctaaact gacgtaatgg gactaaagtg taaaaaatcc cgccaaaccc aacacacacc 34560 ccgaaactgc gtcaccaggg aaaagtacag tttcacttcc gcaatcccaa caagcgtcac 34620 ttcctctttc tcacggtacg tcacatccca ttaacttaca acgtcatttt cccacggccg 34680 cgccgcccct tttaaccgtt aaccccacag ccaatcacca cacggcccac actttttaaa 34740 atcacctcat ttacatattg gcaccattcc atctataagg tatattattg atgatg 34796 2 2550 DNA Artificial Sequence gag expression cassette 2 ccattgcata cgttgtatcc atatcataat atgtacattt atattggctc atgtccaaca 60 ttaccgccat gttgacattg attattgact agttattaat agtaatcaat tacggggtca 120 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 180 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 240 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 300 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 360 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 420 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat 480 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat 540 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc 600 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt 660 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga 720 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt 780 gccaagagtg agatctacca tgggtgctag ggcttctgtg ctgtctggtg gtgagctgga 840 caagtgggag aagatcaggc tgaggcctgg tggcaagaag aagtacaagc taaagcacat 900 tgtgtgggcc tccagggagc tggagaggtt tgctgtgaac cctggcctgc tggagacctc 960 tgaggggtgc aggcagatcc tgggccagct ccagccctcc ctgcaaacag gctctgagga 1020 gctgaggtcc ctgtacaaca cagtggctac cctgtactgt gtgcaccaga agattgatgt 1080 gaaggacacc aaggaggccc tggagaagat tgaggaggag cagaacaagt ccaagaagaa 1140 ggcccagcag gctgctgctg gcacaggcaa ctccagccag gtgtcccaga actaccccat 1200 tgtgcagaac ctccagggcc agatggtgca ccaggccatc tccccccgga ccctgaatgc 1260 ctgggtgaag gtggtggagg agaaggcctt ctcccctgag gtgatcccca tgttctctgc 1320 cctgtctgag ggtgccaccc cccaggacct gaacaccatg ctgaacacag tggggggcca 1380 tcaggctgcc atgcagatgc tgaaggagac catcaatgag gaggctgctg agtgggacag 1440 gctgcatcct gtgcacgctg gccccattgc ccccggccag atgagggagc ccaggggctc 1500 tgacattgct ggcaccacct ccaccctcca ggagcagatt ggctggatga ccaacaaccc 1560 ccccatccct gtgggggaaa tctacaagag gtggatcatc ctgggcctga acaagattgt 1620 gaggatgtac tcccccacct ccatcctgga catcaggcag ggccccaagg agcccttcag 1680 ggactatgtg gacaggttct acaagaccct gagggctgag caggcctccc aggaggtgaa 1740 gaactggatg acagagaccc tgctggtgca gaatgccaac cctgactgca agaccatcct 1800 gaaggccctg ggccctgctg ccaccctgga ggagatgatg acagcctgcc agggggtggg 1860 gggccctggt cacaaggcca gggtgctggc tgaggccatg tcccaggtga ccaactccgc 1920 caccatcatg atgcagaggg gcaacttcag gaaccagagg aagacagtga agtgcttcaa 1980 ctgtggcaag gtgggccaca ttgccaagaa ctgtagggcc cccaggaaga agggctgctg 2040 gaagtgtggc aaggagggcc accagatgaa ggactgcaat gagaggcagg ccaacttcct 2100 gggcaaaatc tggccctccc acaagggcag gcctggcaac ttcctccagt ccaggcctga 2160 gcccacagcc cctcccgagg agtccttcag gtttggggag gagaagacca cccccagcca 2220 gaagcaggag cccattgaca aggagctgta ccccctggcc tccctgaggt ccctgtttgg 2280 caacgacccc tcctcccagt aaaataaagc ccgggcagat ctgatctgct gtgccttcta 2340 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca 2400 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc 2460 attctattct ggggggtggg gtggggcagc acagcaaggg ggaggattgg gaagacaata 2520 gcaggcatgc tggggatgcg gtgggctcta 2550 3 2645 DNA Artificial Sequence SEAP expression cassette 3 ccattgcata cgttgtatcc atatcataat atgtacattt atattggctc atgtccaaca 60 ttaccgccat gttgacattg attattgact agttattaat agtaatcaat tacggggtca 120 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 180 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 240 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 300 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 360 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 420 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat 480 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat 540 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc 600 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt 660 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga 720 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt 780 gccaagagtg agatcgatct aagtaagctt cctgcatgct gctgctgctg ctgctgctgg 840 gcctgaggct acagctctcc ctgggcatca tcccagttga ggaggagaac ccggacttct 900 ggaaccgcga ggcagccgag gccctgggtg ccgccaagaa gctgcagcct gcacagacag 960 ccgccaagaa cctcatcatc ttcctgggcg atgggatggg ggtgtctacg gtgacagctg 1020 ccaggatcct aaaagggcag aagaaggaca aactggggcc tgagataccc ctggccatgg 1080 accgcttccc atatgtggct ctgtccaaga catacaatgt agacaaacat gtgccagaca 1140 gtggagccac agccacggcc tacctgtgcg gggtcaaggg caacttccag accattggct 1200 tgagtgcagc cgcccgcttt aaccagtgca acacgacacg cggcaacgag gtcatctccg 1260 tgatgaatcg ggccaagaaa gcagggaagt cagtgggagt ggtaaccacc acacgagtgc 1320 agcacgcctc gccagccggc acctacgccc acacggtgaa ccgcaactgg tactcggacg 1380 ccgacgtgcc tgcctccgcc cgccaggagg ggtgccagga catcgctacg cagctcatct 1440 ccaacatgga cattgacgtg atcctaggtg gaggccgaaa gtacatgttt cgcatgggaa 1500 ccccagaccc tgagtaccca gatgactaca gccaaggtgg gaccaggctg gacgggaaga 1560 atctggtgca ggaatggctg gcgaagcgcc agggtgcccg gtatgtgtgg aaccgcactg 1620 agctcatgca ggcttccctg gacccgtctg tgacccatct catgggtctc tttgagcctg 1680 gagacatgaa atacgagatc caccgagact ccacactgga cccctccctg atggagatga 1740 cagaggctgc cctgcgcctg ctgagcagga acccccgcgg cttcttcctc ttcgtggagg 1800 gtggtcgcat cgaccatggt catcatgaaa gcagggctta ccgggcactg actgagacga 1860 tcatgttcga cgacgccatt gagagggcgg gccagctcac cagcgaggag gacacgctga 1920 gcctcgtcac tgccgaccac tcccacgtct tctccttcgg aggctacccc ctgcgaggga 1980 gctccatctt cgggctggcc cctggcaagg cccgggacag gaaggcctac acggtcctcc 2040 tatacggaaa cggtccaggc tatgtgctca aggacggcgc ccggccggat gttaccgaga 2100 gcgagagcgg gagccccgag tatcggcagc agtcagcagt gcccctggac gaagagaccc 2160 acgcaggcga ggacgtggcg gtgttcgcgc gcggcccgca ggcgcacctg gttcacggcg 2220 tgcaggagca gaccttcata gcgcacgtca tggccttcgc cgcctgcctg gagccctaca 2280 ccgcctgcga cctggcgccc cccgccggca ccaccgacgc cgcgcacccg ggttaacccg 2340 tggtccccgc gttgcttcct ctgctggccg ggacatcagg tggcccccgc tgaattggaa 2400 tcgatcagaa ttgatctgat ctgctgtgcc ttctagttgc cagccatctg ttgtttgccc 2460 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa 2520 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg 2580 gcagcacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg 2640 ctcta 2645 4 49 DNA Artificial Sequence short synthetic polyA signal 4 aataaaagat ctttattttc attagatctg tgtgttggtt ttttgtgtg 49 5 35167 DNA adenovirus serotype 24 5 catcatcaat aatatacccc acaaagtaaa caaaagttaa catgcaaatg agcttttgaa 60 tttagggcgg ggccagcgct gattggacga gagaagatga tgcaaatgac gtcacgacgc 120 acggctaacg gtcgccgcgg aggcgtggcc tagcccggaa gcaagtcgcg gggctgatga 180 cgtataaaaa agcggacttt agacccggaa acggccgatt ttcccgcggc cacgcccgga 240 tatgaggtaa ttctgggcgg atgcaagtaa aattaggtca ttttggcgcg aaaactgaat 300 gaggaagtga aaagtgaaaa ataccggtcc cgcccagggc ggaatattta ccgagggccg 360 agagactttg accgattacg tgggggtttc gattgcggtg ttttttcgcg aatttccgcg 420 tccgtgtcaa agtccggtgt ttatgtcaca gatcagctga tccacagggt atttaaacca 480 gtcgagcccg tcaagaggcc actcttgagt gccagcgagt agagatttct ctgagctccg 540 ctcccagagt ctgagaaaaa tgagacacct gcgcctcctt tcttcaactg tgcctattga 600 catggccgca ttattgctgg aggattatgt gagtacaata ttggaggacg aactgcatcc 660 atctccattt gagctgggac ctacacttca ggacctatat gatttggagg tagatgccca 720 tgatgacgac ccgaacgaag aggctgtgaa tttaatattt ccagaatctc tgattcttca 780 ggctgacata gccagcgaag ctgtacctac accacttcat acaccgactc tgtcacccat 840 acctgaattg gaagaggagg acgagctaga cctccgatgt tatgaggaag gttttcctcc 900 cagcgattca gaggacgaac agggtgagca gagcatggct ctaatctcaa aatatgcttg 960 tgtggttgtg gaagagcatt ttgtgttgga caatcctgag gtgcccgggc aaggctgtag 1020 atcctgccag taccaccggg ataagaccgg agacacgaac gcctcctgcg ctctgtgtta 1080 catgaaaaag aacttcagct ttatttacag taagtggagt gaatgtgaga gagactgagt 1140 gcttaacaca taactgggta atgcttaaac agctgtgcta agtgtggttt atttttgttt 1200 ctaggtccgg tgtcagagga tgagtcatca ccctcagaag aagaccaccc gtgtccccct 1260 gagctgtcag gcgaaacgcc cctgcaagtg cacagaccca ccccagtcag acccagtggc 1320 gagaggcgag cagctgttga aaaaattgag gacttgttac atgacatggg tggggatgaa 1380 cctttggacc tgagcttgaa acgccccagg aactaggctc agctgtgctt agtcatgtgt 1440 aaataaagtt gtacaataaa agtatatgtg acgcatgcaa ggtgtggttt atgactcatg 1500 ggcgtggctt agtcctatat aagtggcaac acctgggcac tggggcacag accttcaggg 1560 agttcctgat ggatgtgtgg actatccttg cagactttag caagacacgc cggcttgtag 1620 aggatagttc agacgggtgc tccgggttct ggagacactg gtttggaact cctctatctc 1680 gtctggtgta cacagttaag aaggattata acgaggaatt tgaaaatctt tttgctgatt 1740 gctctggcct gctagattct ctaaatctcg gccaccagtc ccttttccag gaaagggtac 1800 tccacagcct tgatttttca agcccagggc gcactacagc cggggttgct tttgtggttt 1860 ttctggttga caaatggagc cagaacaccc aactgagcag gggctacatt ctggacttcg 1920 cagccatgca cctgtggagg gcatgggtga ggcagcgggg acagagaatc ttgaactact 1980 ggcttataca gccagcagct ccgggtcttc ttcgtctaca cagacaaaca tccatgttgg 2040 aggaagaaat gaggcaggcc atggacgaga acccgaggag cggcctggac cctccgtcgg 2100 aagaggagct ggattgaatc aggtatccag cctgtaccca gagcttagca gggtgctgac 2160 atccatggcc aggggagtga agagggagag gagcgatggg ggcaataccg ggatgatgac 2220 cgagctgacg gccagcctga tgaatcgcaa gcgtccagag cgcattacct ggcacgagct 2280 acagatggag tgtagggatg aggtgggcct gatgcaggat aaatatggcc tggagcagat 2340 aaaaacccac tggttgaacc cagatgagga ttgggaggag gccattaaga aatatgccaa 2400 gatagccctg cgcccagatt gcaagtacag ggtgaccaag acggtgaata tcagacatgc 2460 ctgctacatc tcggggaacg gggcagaggt ggtcatcgat accctggaca aggccgcctt 2520 caggtgttgc atgatgggaa tgagagccgg agtgatgaat atgaattcca tgattttcat 2580 gaacatgaag ttcaatggag agaagtttaa tggggtgatg ttcatggcca acagtcacat 2640 gaccctgcac ggctgcagtt tcttcggctt caacaatatg tgcgcagagg tctggggcgc 2700 tgctaagatc aggggatgta agttttatgg ctgctggatg ggcgtggtcg gaagacccaa 2760 gagcgagatg tctgtgaagc agtgtgtgtt tgagaaatgc tacctgggag tctctaccga 2820 gggcaatgct agagtgagac attgctcttc cctggagacg ggctgcttct gcctggtgaa 2880 gggcacagcc tctctgaagc ataatatggt gaagggctgc acggatgagc gcatgtacaa 2940 catgctgaca tgcgactcgg gggtctgcca tatcctgaag aacatccatg tgacctccca 3000 cccccggaag aagtggccag tgtttgagaa taacctactg atcaagtgcc acatgcacct 3060 gggcgccaga aggggcacct tccagccgta ccagtgcaac tttagccaga ccaagctgct 3120 gctggagaac gatgccttct ccagggtgaa cctgaacggc atctttgaca tggatgtctc 3180 ggtgtacaag atcctgagat acgatgagac caagtccagg gtgcgcgctt gcgagtgcgg 3240 gggcagacac accaggatgc aaccagtggc cctggatgtg accgaggagc tgaggcccga 3300 ccacctggtg atggcttgta ccgggaccga gttcagctcc agtggggagg acacagatta 3360 gaggtaggtt gagtattagt gggcgtggct aaggtgacta taaaggcggg tgtcttacga 3420 gggtcttttt gcttttctgc agacatcatg aacgggactg gcggggcctt cgaagggggg 3480 ctttttagcc cttatttgac aacccgcctg ccgggatggg ccggagttcg tcagaatgtg 3540 atgggatcga cggtggacgg gcgtccagtg cttccagcaa attcctcgac catgacctac 3600 gcgaccgtgg ggaactcgtc gctcgacagc accgccgcag ccgcggcagc cgcagccgcc 3660 atgacagcga cgagactggc ttcgagctac atgcccagca gcagcagtag cccctctgtg 3720 cccagttcca tcatcgccga ggagaaactg ctggccctgc tggccgagct ggaagccctg 3780 agccgccagc tggccgccct gacccagcag gtgtccgagc tccgcgaaca gcagcagcag 3840 caaaataaat gattcaataa acacagattc tgattcaaac agcaaagcat ctttattatt 3900 tattttttcg cgcgcggtag gccctggtcc acctctcccg atcattgaga gtgcggtgga 3960 ttttttccag gacccggtag aggtgggatt ggatgttgag gtacatgggc atgagcccgt 4020 cccgggggtg gaggtagcac cactgcatgg cctcgtgctc tggggtcgtg ttgtagatga 4080 tccagtcata gcaggggcgc tgggcgtggt gctggatgat gtccttgagg aggagactga 4140 tggccacggg gagccccttg gtgtaggtgt tggcgaagcg gttgagctgg gagggatgca 4200 tgcgggggga gatgatgtgg agtttggcct ggatcttgag gttggcgatg ttgccaccca 4260 gatcccgcct ggggttcatg ttgtgcagga ccaccagaac ggtgtagccc gtgcacttgg 4320 ggaacttgtc atgcaacttg gaagggaatg cgtgaaagaa tttggagacg cccttgtgcc 4380 cacccaggtt ttccatgcac tcatccatga tgatggcgat gggcccgtgg gctgcggctt 4440 tggcaaagac gtttctgggg tcagagacat cgtaattatg ctcctgggtg agatcatcat 4500 aagacatttt aatgaatttg gggcggaggg tgccagattg ggggacaatg gttccctcgg 4560 gccccggggc gaagttcccc tcacatattt gcatctccca ggctttcatc tcggaggggg 4620 ggatcatgtc cacctgcggg gcgatgaaaa aaacggtttc cggggcgggg gtgatgagct 4680 gcgaggagag caggtttctc aacagctggg acttgccgca cccggtcggg ccgtagatga 4740 ccccgatgac gggttgcagg tggtagttca aggacatgca gctgccgtcg tcccggagga 4800 ggggggccac ctcgttgagc atgtctctga cttggaggtt ttcccggacg agctcgccga 4860 ggaggcggtc cccgcccagc gagagcagct cttgcaggga agcaaagttt ttcaggggct 4920 tgagcccgtc ggccatgggc atcttggcga gggtctgcga gaggagttcg aggcggtccc 4980 agagctcggt gacgtgctct acggcatctc gatccagcag acttcctcgt ttcgggggtt 5040 gggacgactg cgactgtagg gcacgagacg atgggcgtcc agcgctgcca gcgtcatgtc 5100 cttccagggt ctcagtgtcc gcgtgagcgt ggtctccgtc acggtgaagg ggtgggcccc 5160 gggctgtgcg cttgcaaggg tgcgcttgag actcatcctg ctggtgctga aacgggcacg 5220 gtcttcgccc tgcgcgtcgg cgagatagca gttgaccatg agctcgtagt tgagggcctc 5280 ggcggcgtgg cccttggcgc ggagcttgcc cttggaagag cgcccgcagg cgggacagag 5340 gagggattgc agggcgtaga gcttgggtgc gagaaagacg gactcggggg cgaaagcatc 5400 cgctccgcag tgggcgcaga cggtctcgca ctcgaccagc caggtgagct cgggctgctc 5460 ggggtcaaaa accagttttc ccccgttctt tttgatgcgc ttcttacctc gcgtctccat 5520 gagtctgtgt ccgcgctcgg tgacaaacag gctgtctgtg tccccgtaga cggacttgat 5580 gggcctgtcc tgcaggggcg tcccgcggtc ctcctcgtag agaaactcgg accactctga 5640 gacgaaggcg cgcgtccacg ccaagacaaa ggaggccacg tgcgaggggt agcggtcgtt 5700 gtccaccagg gggtccacct tttccacggt atgcagacac atgtccccct cctccgcatc 5760 caagaaggtg attggcttgt aggtgtaggc cacgtgaccc ggggtccccg acgggggggt 5820 ataaaagggg gcgggtctgt gctcgtcctc actctcttcc gcgtcgctgt ccacgagcgc 5880 cagctgttgg ggtaggtatt ccctttcgag agcgggcatg acctcggcac tcaggttgtc 5940 agtttctaga aacgaggagg atttgatgtt ggcttgccct gccgcaatgc tttttaggag 6000 actttcatcc atctggtcag aaaagactat ttttttattg tcaagcttgg tggcgaagga 6060 gccatagagg gcgttggaga gaagcttggc gatggatctc atggtctgat ttttgtcacg 6120 gtcggctcgc tccttggccg cgatgttgag ctggacatac tcgcgcgcga cgcacttcca 6180 ttcggggaag acggtggtgc gctcgtcggg cacgatcctg acgcgccagc cgcggttatg 6240 cagggtgacc agatccacgc tggtggccac ctcgccgcgc aggggctcgt tggtccagca 6300 gaggcgtccg cccttgcgcg agcagaacgg gggcagcaca tcaagcagat gctcgtcagg 6360 ggggtccgca tcgatggtga agatgcccgg acagagttcc ttgtcaaaat aatcgatttt 6420 tgaggatgca tcatccaagg ccatctgcca ctcgcgggcg gccagcgctc gctcgtaggg 6480 gttgaggggc ggaccccagg gcatgggatg cgtcagggcg gaggcgtaca tgccgcagat 6540 gtcgtagaca tagatgggct ccgagaggat gccgatgtag gtgggataac agcgcccccc 6600 gcggatgctg gcgcgcacgt agtcatacaa ctcgtgcgag ggggccaaga aggcggggcc 6660 gagattggtg cgctggggct gctcggcgcg gaagacgatc tggcgaaaga tggcatgcga 6720 gttggaggag atggtgggcc gttggaagat gttaaagtgg gcatgaggca gacgaaccga 6780 gtcgcggatg aagtgcgcgt aggagtcttg cagcttggcg acgagctcgg cggtgacgag 6840 gacgtccatg gcgcagtagt ccagcgtttc gcggatgatg tcataacccg cctctccttt 6900 cttctcccat agctcgcggt tgagggcgta ctcctcgtca tccttccagt actcccggag 6960 cgggaatcct cgatcgtccg cacggtaaga gcccagcatg tagaaatggt tcacggcctt 7020 gtagggacag cagcccttct ccacggggag ggcgtaagct tgagcggcct tgcggagcga 7080 ggtgtgcgtc agggcgaagg tatccctgac catgactttc aagaactggt acttgaaatc 7140 cgagtcgtcg cagccgccgt gctcccagag ctcgaaatcg gtgcgcttct tcgagagggg 7200 gttaggcaga gcgaaagtga cgtcattgaa gagaatcttg cctgcccgcg gcatgaaatt 7260 gcgggtgatg cggaaagggc ccgggacgga ggctcggttg ttgatgacct gggcggcgag 7320 gacgatctcg tcgaagccgt tgatgttgtg cccgacgatg tagagttcca tgaatcgcgg 7380 gcggccttta atgtgcggca gctttttgag ctcctcgtag gtgaggtcct cggggcaatg 7440 cagtccgtgc tgctcgagcg cccactcctg gagatgtggg ttggcttgca tgaatgaagc 7500 ccagagctcg cgggccataa gggtctggag ctcgtcgcga aagaggcgga actgctggcc 7560 cacggccatc ttttctgggg tgacgcagta gaaagtaagg gggtcccgct cccagcgatc 7620 ccagcgtaag cgcacggcta gatcgcgagc gagggcgacc agctctgggt cccccgagaa 7680 tttcataacc agcataaagg ggacgagctg cttgccgaag gaccccatcc aggtgtaggt 7740 ttctacatcg taggtgacaa agagccgctc cgtgcgagga tgagagccga ttgggaagaa 7800 ctggatttcc tgccaccagt tggacgagtg gctgttgatg tgatgaaagt agaaatcccg 7860 ccggcgaacc gagcactcgt gctgatgctt gtaaaagcgt ccgcagtact cgcagcgctg 7920 cacgggctgt acctcatcca cgagatacac agcgcgtccc ttgaggagga acttcaggag 7980 tggcggccct ggctggtggt tttcatgttc gcctgcgtgg gactcaccct ggggctcctc 8040 gaggacggag aggctgacga gcccgcgcgg gagccaggtc cagatctcgg cgcggcgggg 8100 gcggagagcg aagacgaggg cgcgcagttg ggagctgtcc atggtgtcgc ggagatccag 8160 gtccgggggc agggttctga ggttgacctc gtagaggcgg gtgagggcgt gcttgagatg 8220 cagatggtac ttgatctcca cgggtgagtt ggtggctgtg tccacgcatt gcatgagccc 8280 gtagctgcgc ggggccacga ccgtgccgcg gtgcgctttt agaagcggtg tcgcggacgc 8340 gctcccggcg gcagcggcgg ttccggcccc gcgggcaggg gcggcagagg cacgtcggcg 8400 tggcgctcgg gcaggtcccg gtgctgcgcc ctgagagcgc tggcgtgcgc gacgacgcgg 8460 cggttgacat cctggatctg ccgcctctgc gtgaagacca ccggccccgt gactttgaac 8520 ctgaaagaca gttcaacaga atcaatctcg gcgtcattga cggcggcctg acgcaggatc 8580 tcttgcacgt cgcccgagtt gtcctggtag gcgatctcgg acatgaactg ctcgatctcc 8640 tcctcctgga gatcgccgcg gcccgcgcgc tccacggtgg cggcgaggtc attggagatg 8700 cgacccatga gctgcgagaa ggcgcccagg ccgctctcat tccagacgcg gctgtagacc 8760 acgtccccgt cggcgtcgcg cgcgcgcatg accacctgcg cgaggttgag ctccacgtgc 8820 cgcgtgaaga cggcgtagtt gcgcaggcgc tggaagaggt agtttagggt ggtggcgatg 8880 tgctcggtga cgaagaagta catgatccag cggcgcaggg gcatctcgct gatgtcgccg 8940 atggcctcca gcctttccat ggcctcgtag aaatccacag cgaagttgaa aaactgggcg 9000 ttgcgggccg agaccgtgag ctcgtcctcc aggagcctga tgagttcggc gatggtggcg 9060 cgcacctcgc gctcgaaatc cccgggggcc tcctcctctt cctcttcttc catgacgacc 9120 tcttcttcta tttcttcctc tgggggcggt ggtggtggcg gggcccgacg acgacggcga 9180 cgcaccggga gacggtcgac gaagcgctcg atcatctccc cgcggcggcg acgcatggtt 9240 tcggtgacgg cgcgaccccg ttcgcgagga cgcagcgtga agacgccgcc ggtcatctcc 9300 cggtaatggg gcgggtcccc gttgggcagc gagagggcgc tgacgatgca tcttatcaat 9360 tgcggtgtag gggacgtgag cgcgtcgaga tcgaccggat cggagaatct ttcgaggaaa 9420 gcgtctagcc aatcgcagtc gcaaggtaag ctcaaacacg tagcagccct gtggacgctg 9480 ttagaattgc ggttgctgat gatgtaattg aagtaggcgt ttttaaggcg gcggatggtg 9540 gcgaggagga ccaggtcctt gggtcccgct tgctggatgc gaagccgctc ggccatgccc 9600 caggcctggc cctgacaccg gctcaggttc ttgtagtagt catgcatgag cctctcaatg 9660 tcatcactgg cggaggcgga gtcttccatg cgggtgaccc cgacgcccct gagcggctgc 9720 acgagcgcca ggtcggcgac gacgcgctcg gcgaggatgg cctgttgcac gcgggtgagg 9780 gtgtcctgga agtcgtccat gtcgacgaag cggtggtagg ccccggtgtt gatggtgtag 9840 gtgcagttgg ccatgagcga ccagttgacg gtctgcaggc cgggttgcac gacctctgag 9900 tacctgagcc gcgagaaggc gcgcgagtcg aagacatagt cgttgcaggt gcgcacgagg 9960 tactggtatc caactaggaa gtgcggcggc ggctggcggt agagcggcca gcgctgggtg 10020 gccggcgcgc ccggggccag gtcctcgagc atgaggcggt ggtagccgta gaggtagcgg 10080 gacatccagg tgatgccggc ggcggtggtg gaggcgcgcg ggaactcgcg gacgcggttc 10140 cagatgttgc gcagcggcag gaaatagtcc atggtcggca cggtctggcc ggtgagacgc 10200 gcgcagtcat tgacgctcta gaggcaaaaa cgaaagcggt tgagcgggct cttcctccgt 10260 agcctggcgg aacgcaaacg ggttaggccg cgtgtgtacc ccggttcgag tcccctcgaa 10320 tcaggctgga gccgcgacta acgtggtatt ggcactcccg tctcgacccg agcccgatag 10380 ccgccaggat acggcggaga gccctttttg ccgaccgagg ggagtcgcta gacttgaaag 10440 cggccgaaaa ccccgccggg tagtggctcg cgcccgtagt ctggagaagc tttgccaggg 10500 ttgagtcgcg gcagaacccg gttcgcggac ggccgcggcg agcgggactt ggtcaccccg 10560 ccgatttaaa gacccacagc cagccgactt ctccagttac gggagcgagc cccctttttt 10620 ctttttgcca gatgcatccc gtcctgcgcc aaatgcgtcc caccccccct ccggcgacca 10680 ccgcgaccgc ggccgtagca ggcgccggcg ctgtagcccc gccacagcag acagagatgg 10740 acttggaaga gggcgaaggg ctggcgagac tgggggcgcc gtccccggag cgacaccccc 10800 gcgtgcagct gcagaaggac gtgcgcccgg cgtacgtgcc tgcgcagaac ctgttcaggg 10860 accgcagcgg ggaggagccc gaggagatgc gcgactgccg ttttcgggcg ggcagggagc 10920 tgcgcgaggg cctggaccgc cagcgcgtgc tgcgcgacga ggatttcgag ccgaacgagc 10980 agacggggat cagccccgcg cgcgcgcacg tggcggcggc caacctggtg acggcctacg 11040 agcagacggt gaagcaggag cgcaacttcc aaaagagttt caacaaccat gtgcgcacgc 11100 taatcgcgcg cgaggaggtg gccctgggct tgatgcacct gtgggacctg gcggaggcca 11160 tcgtgcagaa cccggacagc aagcctctga cggcgcagct gttcctggtg gtgcagcaca 11220 gcagggacaa cgaggcgttc agggaggcgc tgctaaacat cgccgagccc gagggccgct 11280 ggctgctgga gctgatcaac atcttgcaga gcatcgtagt gcaggagcgc agcctgagcc 11340 tggccgagaa ggtggcggct atcaactact cggtgctgag cctgggcaag ttttacgcgc 11400 gcaagattta caagacgccg tacgtgccca tagacaagga ggtgaagata gacagctttt 11460 acatgcgcat ggcgctcaag gtgctgacgc tgagcgacga cctgggcgtg taccgcaacg 11520 accgcatcca caaggccgtg agcgcgagcc ggcggcgcga gctgagcgac cgcgagctga 11580 tgctgagtct gcgccgggcg ctggtagggg gcgccgccgg cggtgaggag tcctacttcg 11640 acatgggggc ggacctgcat tggcagccga gccggcgcgc cttggaggcc gcctacggtc 11700 cagaggactt ggatgaggat gaggaagagg aggaggatgc acccgctgcg gggtactgac 11760 gcctccgtga tgtgttttta gatgcagcaa gccccggacc ccgccataag ggcggcgctg 11820 caaagccagc cgtccggtct agcatcggac gactgggagg ccgcgatgca acgcatcatg 11880 gccctgacga cccgcaaccc cgagtccttt agacaacagc cgcaggccaa cagactctcg 11940 gccattctgg aggcggtggt cccctctcgg accaacccca cgcacgagaa ggtgctggcg 12000 atcgtgaacg cgctggcgga gaacaaggcc atccgtcccg acgaggccgg gctggtgtac 12060 aacgccctgc tggagcgcgt gggccgctac aacagcacaa acgtgcagtc caacctggac 12120 cggctggtga cggacgtgcg cgaggccgtg gcgcagcgcg agcggttcaa gaacgagggc 12180 ctgggctcgt tggtggcgct gaacgccttc ctggcgacgc agccggcgaa cgtgccgcgc 12240 gggcaggacg attacaccaa ctttatcagc gcgctgcggc tgatggtgac cgaggtgccc 12300 cagagcgagg tgtaccagtc gggcccagac tactttttcc agacgagccg gcagggcttg 12360 cagacggtga acctaagcca ggctttcaag aatctgcgcg ggctgtgggg cgtgcaggcg 12420 cccgtgggcg accggtcgac ggtgagcagc ttgctaacgc ccaactcgcg gctgctgctg 12480 ctgctgatcg cgcccttcac cgacagcggc agcgtgaacc gcaactcgta cctgggccac 12540 ctgctgacgc tttaccgcga ggccataggc caggcgcagg tggacgagca gaccttccag 12600 gagatcacta gcgtgagccg cgcgctgggt cagaacgaca ccgacagtct gagagccacc 12660 ctgaacttct tgctgacaaa tagacagcag aagattccgg cgcagtacgc gctgtcggcc 12720 gaggaggagc gcatcctgag atatgtgcag cagagcgtag ggcttttcct gatgcaggag 12780 ggggccaccc ccagcgccgc gctggacatg accgcgcgca acatggaacc tagcatgtac 12840 gccgccaacc ggccgttcat caataagctg atggactacc tgcaccgcgc ggctgccatg 12900 aactcggact actttactaa tgctatacta aacccgcact ggctcccgcc gccggggttc 12960 tacacgggcg agtacgacat gcccgacccc aacgatgggt tcctgtggga cgacgtggac 13020 agcgcggtgt tctccccgac cttgcaaaag cgccaggagg cggtacgcac gcccgcgagc 13080 gagggcgcgg tgggtcggag cccctttcct agcttaggga gtttgcatag cttgccgggc 13140 tcggtgaaca gcggcagggt gagccggccg cgcttgctgg gcgaggacga gtacctgaac 13200 gactcgctgc tgcagccgcc gcgggtcaag aacgccatgg ccaataacgg gatagagagt 13260 ctggtggaca aactgaaccg ctggaagacc tacgctcagg accataggga tgcgcccgcg 13320 ccgcggcgac agcgccacga ccggcagcgg ggcctggtgt gggacgacga ggactcggcc 13380 gacgatagca gcgtgttgga cttgggcggg agcggtgggg ccaacccgtt cgcgcatctg 13440 cagcccagac tggggcgacg gatgttttga atgaaataaa actcaccaag gccatagcgt 13500 gcgttctctt ccttgttaga gatgaggcgc gcggtggtgt cttcctctcc tcctccctcg 13560 tacgagagcg tgatggcgca ggcaaccctg gaggttccgt ttgtgcctcc gcggtatatg 13620 gctcctacgg agggcagaaa cagcattcgt tactcggaac tggctccgca gtacgacacc 13680 actcgcgtgt acttggtgga caacaagtcg gcggacatcg cttccctgaa ctaccaaaac 13740 gaccacagca acttcctgac cacggtggtg cagaacaacg atttcacccc cgccgaggcc 13800 agcacgcaga cgataaattt tgacgagcgg tcgcggtggg gcggtgattt gaagaccatt 13860 ctgcacacca acatgcccaa tgtgaacgag tacatgttca ccagcaagtt taaggcgcgg 13920 gtgatggtgg ctaggaaggt ggtagatcag aatgatagga gcaaggatga gttaaaatat 13980 gagtggtttg agtttaccct gcccgagggc aacttttccg agaccatgac catagacctg 14040 atgaacaacg ccatcttgga aaactacttg caagtggggc ggcaaaatgg cgtgctggag 14100 agcgatatcg gagtcaagtt tgacagcagg aatttcaagc tgggctggga cccggtaacc 14160 aagctggtga tgcctggggt ctacacctac gaggccttcc acccggacgt tgtgctgctg 14220 ccgggctgcg gggtggactt caccgagagc cgcctgagca acctcctggg cattcgcaag 14280 aagcaacctt tccaagaggg cttcaggatc atgtatgagg atctcgaggg tggtaacatc 14340 cccgccctcc tggatgtcaa gcaatatttg gatagtaaaa agaagcttga ggaggcaaca 14400 cagaatgcaa ccagggctgc tggagatatc agaggagaca gtcatattcc aagagctgtg 14460 gaacaagcgg ctgaaaagga tctggtcatt gtaccagtaa cacaagatga aagtaagaga 14520 agctataatg tcatagatgg cacccatgac accctctacc gaagttggta cctgtcctat 14580 acctacgggg accccgagaa gggggtgcag tcgtggacgc tgctcaccac cccggacgtc 14640 acctgcggcg cggagcaagt ctactggtcg ctgccggacc tcatgcaaga ccccgtcacc 14700 ttccgctcta cccagcaagt cagcaactac cccgtggttg gcgccgagct catgcccttc 14760 cgcgccaaga gcttttacaa cgacctcgcc gtctactccc agctcatccg cagctacacc 14820 tccctcaccc acgtcttcaa ccgcttcccc gacaaccaga tcctctgccg tccgcccgcg 14880 cccaccatca ccacggtcag tgaaaacgtg cctgctctca cagatcacgg gacgctaccg 14940 ctgcgcagca gtatccgcgg agtccagcga gtgaccgtca ctgacgcccg tcgccgcacc 15000 tgtccctacg tctacaaggc cctgggcata gtcgcgccgc gcgtgctttc cagtcgcacc 15060 ttctaaaaaa tgtctattct catctcgccc agcaataaca ccggctgggg tcttactagg 15120 cccagcacca tgtacggagg agccaagaag cgctcccagc agcaccccgt ccgcgtccgc 15180 ggccacttcc gcgctccctg gggcgcttac aagcgcgggc ggacttctac cgccgccgtg 15240 cgcaccaccg tcgacgacgt catcgactcg gtggtcgccg acgcgcgcaa ctataccccc 15300 gccccctcca ccgtggacgc ggtcatcgac agcgtggtgg ccgacgcgcg cgactatgcc 15360 agacgcaaga gccggcggcg acggatcgcc aggcgccacc ggagtacgcc cgccatgcgc 15420 gccgcccggg ctctgctgcg ccgcgccaga cgcacgggcc gccgggccat gatgcgagcc 15480 gcgcgccgcg ccgccactgc accccccgca ggcaggactc gcagacgagc ggccgccgcc 15540 gctgccgcgg ccatttctag catgaccaga cccaggcgcg gaaacgtgta ctgggtgcgc 15600 gactccgtca cgggcgtgcg cgtgcccgtg cgcacccgtc ctcctcgtcc ctgatctaat 15660 gcttgtgtcc tcccccgcaa gcgacgatgt caaagcgcaa aatcaaggag gagatgctcc 15720 aggtcgtcgc cccggagatt tacggaccac cccaggcgga ccagaaaccc cgcaaaatca 15780 agcgggttaa aaaaaaggat gaggtggacg agggggcagt agagtttgtg cgcgagttcg 15840 ctccgcggcg gcgcgtaaat tggaaggggc gcagggtgca gcgcgtgttg cggcccggca 15900 cggcggtggt gtttacgccc ggcgagcggt cctcggtcag gagcaagcgt agctatgacg 15960 aggtgtacgg cgacgacgac atcctggacc aggcggcgga gcgggcgggc gagttcgcct 16020 acgggaagcg gtcgcgcgaa gaggagctga tctcgttgcc gctggacgag agcaacccca 16080 cgcctagcct gaagcccgtg accctgcagc aggtgctgcc ccaagcagtg ctgctgccga 16140 gccgcggggt caagcgcgag ggcgagaata tgtacccgac catgcagatc atggtgccca 16200 agcgccggcg cgtggaagaa gtgctggaca ccgtgaaaat ggatgtggag cccgaggtca 16260 aggtgcgccc catcaagcag gtggcgccgg gcctgggcgt gcagaccgtg gacattcaga 16320 tccccaccga catggatgtt gacaaaaaac cctcgaccag catcgaggtg cagaccgacc 16380 cctggctccc agcctccacc gctgccgtct ccacttctac cgccgccacg gctaccgagc 16440 ctcccagaag gcgaagatgg ggccctgcca accggctgat gcccaactac gtattgcatc 16500 cttccattat cccgacgccg ggctatcgcg gcacccggta ctacgccagc cgcaggcgcc 16560 cagccagcaa acgccgccgc cgcaccgcca cccgccgccg tctggccccc gcccgcgtgc 16620 gccgcgtaac cacgcgccgg ggccgctcgc tcgttctgcc caccgtgcgc taccacccca 16680 gcatccttta atccgtgtgc tgtgatactg ttgcagagag atggctctca cttgccgcct 16740 gcgcatcccc gtcccgaatt accgaggaag atcccgccgc aggagaggca tggcaggcag 16800 cggcctcaac cgccgccggc ggcgggccat gcgcaggcgc ctgagtggcg gctttctgcc 16860 cgcgctcatc cccataatcg cggcggccat cggcacgatc ccgggcatag cttccgttgc 16920 gctgcaggcg tcgcagcgcc gttgatgtgc gaataaagcc tctttagact ctgacacacc 16980 tggtcctgta tatttttaga atggaagaca tcaattttgc gtccctggct ccgcggcacg 17040 gcacgcggcc gttcatgggc acctggaacg agatcggcac cagccagctg aacgggggcg 17100 ccttcaattg gagcagtgtc tggagcgggc ttaaaaattt cggctcgacg ctccggacct 17160 atgggaacaa ggcctggaat agtagcacgg ggcagttgtt aagggaaaag ctcaaagacc 17220 agaacttcca gcagaaggtg gtggacggcc tagcctcggg cattaacggg gtggtggaca 17280 tagcaaacca ggccgtgcag cgcgagataa acagccgcct ggacccgcgg ccgcccacgg 17340 tggtggagat ggaagatgca actcctccgc cgcccaaggg cgagaagcgg ccgcggcccg 17400 acgcggagga gacgatcctg caggtggacg agccgccctc gtacgaggag gccgtcaagg 17460 ccggcatgcc caccacgcgt atcatcgcgc cactggccac tggtgtaatg aaacccgcca 17520 cccttgacct gcctccgcca cccacgcccg ctccaccgaa ggcagctccg gttgtgcagc 17580 cccctcctgt ggcgaccgcc gtgcgccgcg tccccgcccg ccgccaggcc cagaactggc 17640 agagcacgct gcacagtatc gtgggcctgg gagtgaaaag tctgaagcgc cgccgatgct 17700 attgagagag aggaaagagg acactaaagg gagagcttaa cttgtatgtg ccttaccgcc 17760 agagaacgcg cgaagatggc taccccctcg atgatgccgc agtgggcgta catgcacatc 17820 gccgggcagg acgcctcgga gtacctgagc ccgggtctgg tgcagtttgc ccgcgccacc 17880 gacacgtact tcagcctggg caacaagttt aggaacccca cggtggctcc cacccacgat 17940 gtgaccacgg accggtccca gcgtctgacg ctgcgctttg tgcccgtgga tcgcgaggac 18000 accacgtact cgtacaaggc gcgcttcact ctggccgtgg gcgacaaccg ggtgctagac 18060 atggccagca cttactttga catccgcggc gtcctggacc gcggtcccag cttcaaaccc 18120 tactcgggca cggcttacaa cagcctggcc cccaaaggcg cccccaactc tagtcagtgg 18180 gaacaagcta aagctaccaa tgccggtcaa aaggaaactc acacatttgg agtagccgct 18240 atgggcggag aagacattac agtgaaaggt cttcaaattg gaactgatga aactaaggaa 18300 gatggagagg atgaaatttt tgcagatcaa acattccagc cagaacctca agtgggagaa 18360 cagaactggc aagaaacgtt tgttttctat ggaggcagag ctcttaagaa agaaaccaaa 18420 atgaagccat gttatggctc ttatgcgaga cccacaaatg aaaagggagg acaggctaaa 18480 tttacacttg atgaaaaagg tcagccaacc aaaattcctg atattacaat ggatttcttt 18540 gatagtccac aagatgatac atcaggtgta actaataagc cagatattgt catgtatgca 18600 gaaaatgtaa atttagaagc tcctgacaca catgtagttt acaaaccagg caaagatgat 18660 tctagttctt ccgctaacct cacacaacag gccatgccta acagaccgaa ctacatcggg 18720 ttcagagaca actttgtggg tcttatgtac tacaatagta ctggcaacat gggtgtgctg 18780 gctggtcagg cctctcagtt gaatgctgtg gtcgacttgc aagacagaaa caccgagctg 18840 tcttaccagc tattgctaga ttctctgggt gacagaacca gatactttag catgtggaat 18900 tctgcagtgg acagctatga ccccgatgtc aggatcattg agaatcacgg tgtggaagat 18960 gaacttccaa actattgctt cccactgaat ggcagtggtt ctaacagcac atacaaaggt 19020 gttaaagctg gaactggaaa caattgggat gacgatgaaa atgttgcaag acaaaatcag 19080 attggcactg gcaacctgtt cgccatggag atcaacctcc aggccaacct atggaagagt 19140 tttctgtact cgaacgtggc cctgtacctg cccgactcct acaagtacac gccggccaac 19200 gtcacgctgc ccaccaacac caacacctac gactacatga acggccgcgt ggtagccccc 19260 tcgctggtgg acgcctacat caacattggc gcccgctggt cgctggaccc catggacaat 19320 gtcaatccct tcaaccacca ccgcaacgcg ggcctgcgct accgctccat gctcctgggc 19380 aacggccgct acgtgccctt ccacatccaa gtgccccaaa agttctttgc catcaagaac 19440 ctgcttctgc tccccggttc ctacacctac gagtggaact tccgcaagga cgtcaacatg 19500 atcctgcaga gttccctcgg caacgacctg cgcgtcgacg gcgcctccgt ccgcttcgac 19560 agcgtcaacc tctacgccac cttcttcccc atggcgcaca acaccgcctc caccctggaa 19620 gccatgctgc gcaacgacac caacgaccag tccttcaacg actacctctc ggccgccaac 19680 atgctctacc ccatcccggc caaggccacc aacgtgccca tctccatccc ctcgcgcaac 19740 tgggccgcct tccgcggctg gagtttcacc cggctcaaga ccaaggaaac tccctccctc 19800 ggctcgggtt tcgaccccta ctttgtctac tcgggctcca tcccctacct cgacgggacc 19860 ttctacctca accacacctt caagaaggtc tccatcatgt tcgactcctc ggtcagctgg 19920 cccggcaacg accggctgct cacgccgaac gagttcgaga tcaagcgcag cgttgacggg 19980 gagggctaca acgtggccca atgcaacatg accaaggact ggttcctcgt ccagatgctc 20040 tcccactaca acatcggcta ccagggcttc cacgtgcccg agggctacaa ggaccgcatg 20100 tactccttct tccgcaactt ccagcccatg agcaggcagg tggtcgatga gatcaactac 20160 aaggactaca aggccgtcac cctacccttc cagcacaaca actcgggctt caccggctac 20220 cttgcgccca ccatgcgcca ggggcagccc taccccgcca acttccccta cccgctcatc 20280 ggctccaccg cagttccctc cgtcacccag aaaaagttcc tctgcgacag ggtcatgtgg 20340 cgcatcccat tctccagcaa ctttatgtcc atgggcgccc tcaccgacct gggtcagaac 20400 atgctctatg ccaactcggc ccacgcgctc gacatgacct ttgaggtgga ccccatggat 20460 gagcccaccc tcctctatct tctcttcgaa gttttcgacg tggtcagagt gcaccagccg 20520 caccgcggcg tcatcgaggc cgtctacctg cgcacgccct tctccgccgg caacgctacc 20580 acttaagcat gagcggctcc agcgaacaag agctcgcggc catcgtgcgc gacctgggat 20640 gcgggcccta ctttttggga acccacgaca agcgcttccc tggcttcctt gccggcgaca 20700 agctggcctg cgccatcgtc aacacggccg gccgcgagac cggaggcgtg cactggctcg 20760 cctttggctg gaatccgcgc tcgcgcacct gctacatgtt cgaccccttt gggttctcgg 20820 accgccggct caagcagatt tacagcttcg agtacgaggc catgctgcgc cgaagcgcgc 20880 ttgcctcctc gcccgaccgc tgtctcagcc tcgagcagtc cacccagacc gtgcaggggc 20940 ccgactccgc cgcctgcgga cttttttgtt gcatgttttt gcatgccttc gtgcactggc 21000 ccgaccgacc catggacgga aaccccacca tgaacttgct gacgggggtg ccaaacggca 21060 tgctacaatc gccacaggtg ctgcccaccc tcaggcgcaa ccaggaggag ctctaccgct 21120 tcctcgcgcg ccactcccct tactttcgat cccaccgcgc cgccatcgaa aacgccaccg 21180 cttttgataa aatgaaacaa ctgcgtgtat ctcaataaac agcactttat tttacatgca 21240 ctggagtata tgcaagttat ttaaaagtcg aaggggttct cgcgctcgtc gttgtgcgcc 21300 gcgctgggga gggccacgtt gcggtactgg tacttgggaa gccacttgaa ctcggggatc 21360 accagtttgg gcactggggt ctcggggaag gtctcgctcc acatgcgccg gctcatctgc 21420 agggcgccca gcatgtccgg gccggagatc ttgaaatcac aattggggcc ggtgctctgc 21480 gcgcgcgagt tgcggtacac ggggttgcag cactggaaca ccattagact ggggtacttc 21540 acactggcaa gcacgctctt gtcgctgatc tgatccttgt ccaggtcctc ggcgttgctc 21600 aggccgaacg gggtcatctt gcacagctgg cggcccagga agggcacgct ctgaggcttg 21660 tggttacact cgcagtgcac gggcatcagc atcatccccg cgccgcgctg catattcggg 21720 tagagggcct tgacgaaggc cgtgatctgc ttgaaagctt gctgggcctt agccccctcg 21780 ctgaaaaaca ggccgcagct cttcccgcta aactggttat tcccgcaccc ggcatcatgc 21840 acgcagcagc gcgcgtcatg gctggtcagt tgcaccacgc tacgtcccca gcggttctgg 21900 gtcaccttgg ccttgctggg ctgctccttc aacgcgcgct gcccgttctc gctggtcaca 21960 tccatctcca ccacgtggtc cttgtggatc atcaccgtcc catgcagaca cttgagctga 22020 ccctcgacat cgcagcagcc atgatcccac agggcgcagc cggtgcactc ccagttctta 22080 tgcgcgatcc cgctgtggct gaagatgtaa ccttgcaaca ggcgacccat gacggtgcta 22140 aatgctttct gggtggtgaa ggtcagttgc agaccgcggg cctcctcgtt catccaggtc 22200 tggcacatct tttggaagat ctcggtctgc tcgggcatga gcttgtaagc atcgcgcagg 22260 ccgctgtcga cgcggtagcg ttccatcagc acgttcatgg tatccatgcc cttctcccag 22320 gacgagacca gaggcagact cagggggttg cgcacgttca ggacaccggg ggtcgcaggc 22380 tcgacgatgc gttttccgtc cttgccttcc ttcaacagaa ccggaggctg gctgaatccc 22440 actcccacga ttacggcatc ttcctggggc atctcttcgt cggggtctac cttggtcaca 22500 tgcttggtct ttctggcttg cttctttttt ggagggctgt ccacggggac cacgtcctcc 22560 tcggaagacc cggagcccac ccgctgatac tttcggcgct tggtgggcag aggaggtggt 22620 ggcggcgagg ggctcctctc ctgctccggc ggatagcgcg ccgacccgtg gccccggggc 22680 ggagtggcct ctcgctccat gaaccggcgc acgtcctgac tgccgccggc cattgtttcc 22740 taggggaaga tggaggagca gccgcgtaag caggagcagg aggaggactt aaccacccac 22800 gagcaaccca aaatcgagca ggacctgggc ttcgaagagc cggctcgtct agaaccccca 22860 caggatgaac aggagcacga gcaagacgca ggccaggagg agaccgacgc tgggctccag 22920 catggctacc tgggaggaga ggaggatgtg ctgctaaaac acttgcagcg ccaatccatc 22980 atcctccggg acgccctggc cgaccggagc gaaacccctc tcagcgtcga ggagctgtgt 23040 cgggcctacg agctcaacct cttctcgccg cgcgtgcccc ccaaacgcca gcccaacggc 23100 acctgcgagc ccaacccgcg tctcaacttc tatcccgtct ttgcggtccc cgaggcccta 23160 gccacctatc acatcttttt caagaaccaa aagatccccg tctcctgccg cgccaaccgc 23220 acccgcgccg acgcgctcct cgctctgggg cccggcgcgc gcatacctga tatcgcttcc 23280 ctggaagagg tgcccaagat cttcgaaggg ctcggtcggg acgagacgcg cgcggcaaac 23340 gctctgaaag aaacagcaga ggaagagggt cacactagcg ccctggtaga gttggaaggc 23400 gacaacgcca ggctggccgt gctcaagcgc agcgtcgagc tcacccactt cgcctacccc 23460 gccgtcaacc tcccgcccaa ggtcatgcgt cgcatcatgg atcagctcat catgccccac 23520 atcgaggccc tcgatgaaag tcaggagcag cgccccgagg acgcccggcc cgtggtcagc 23580 gacgagcagc tcgcgcgttg gctcgggacc cgcgaccccc aggctttgga acagcggcgc 23640 aagctcatgc tggccgtggt cctggtcacc ctcgagctcg aatgcatgcg ccgcttcttc 23700 agcgaccccg agaccctgcg taaggtcgag gagaccctgc actacacttt caggcacggt 23760 ttcgtcaggc aggcctgcaa gatctccaac gtggagctga ccaacctggt ctcatgcctg 23820 gggatcctgc acgagaaccg cctgggacag accgtgctcc actctactct gaagggcgag 23880 gcgcgtcggg actatgtccg cgactgtgta tttctcttta tctgccacac ctggcaagca 23940 gccatgggcg tgtggcagca gtgtctcgag gacgaaaatc tgaaggagct ggacaagctt 24000 cttgctagaa accttaaaaa gctgtggacg ggcttcgacg agcgcaccgt cgcctcggac 24060 ctggccgaga tcgtttttcc agaacgcctg aggcagacgc tgaaaggcgg gctgcccgac 24120 ttcatgagcc agagcatgtt gcaaaactac cgcactttca ttctcgagcg atctgggatg 24180 ctacccgcca cctgcaacgc attcccctcc gactttgtcc cgctgagcta ccgcgagtgt 24240 cccccgccgc tgtggagcca ctgctatctc ttgcagctgg ccaactacat cgcctaccac 24300 tcggacgtga tcgaggacgt gagcggcgag gggcttctcg agtgccactg ccgctgcaac 24360 ctgtgctccc cgcaccgctc cctggtctgc aacccccagc ttctgagcga gacccaggtc 24420 atcggtacct tcgagctgca aggtccgcag gagtccaccg ctccgctgaa actcacgccg 24480 gggttgtgga cttccgcgta cctgcgcaaa tttgtacccg aggactacca cgcccatgaa 24540 ataaagttct tcgaggacca atcgcgccca cagcacgcgg atctcacggc ctgcgtcatc 24600 acccagggcg cgatcctcgc ccaattgcac gccatccaaa aatcccgcca agagtttctt 24660 ctaaaaaagg gtagaggggt ctacctggac ccccagacgg gcgaggtgct caacccgggt 24720 ctcccccagc atgccgagga agaagcagga gccgctagtg gagcagatgg aagaagaatg 24780 ggacagccag gcagaggagg acgaatggga ggaggagaca gaggaggaag aattggaaga 24840 ggtggaagag gagcaggaaa cagagcagcc cgtcgccgca ccatccgcgc cggcagcccc 24900 gccggtcacg gatacaacct ccacagctcc ggccaagcct cctcgtagat gggatcgagt 24960 gaagggtgac ggtaagcacg agcggcaggg ctaccgatca tggagggtcc acaaagccgc 25020 gatcatcgcc tgcttgcaag actgcggggg gaacatcgct ttcgcccgcc gctacctgct 25080 cttccaccgc ggggtgaaca tcccccgcaa cgtgttgcat tactaccgtc accttcacag 25140 ctaagaaaaa gcaagtaaga ggagtcgccg gaggaggcct gaggatcgcg gcgaacgagc 25200 cctcgaccac cagggagctg aggaaccgga tcttccccac tctttatgcc atttttcagc 25260 agagtcgagg tcagcagcaa gaactgaaag taaaaaaccg gtctctgcgc tcgctcaccc 25320 gcagttgctt gtaccacaaa aacgaagatc agctgcagcg cactctcgaa gacgccgagg 25380 ctctgttcca caagtactgc gcgctcactc ttaaagacta aggcgcgccc acccggaaaa 25440 aaggcgggaa ttacctcatc gccaccatga gcaaggagat tcccacccct tacatgtgga 25500 gctatcagcc ccagatgggc ctggccgcgg gcgcctccca ggactactcc acccgcatga 25560 actggctcag tgccggcccc tcgatgatct cacgggtcaa cggggtccgt aaccatcgaa 25620 accagatatt gttggagcag gcggcggtca cctccacgcc cagggcaaag ctcaacccgc 25680 gtaattggcc ctccaccctg gtgtatcagg aaatccccgg gccgactacc gtactacttc 25740 cgcgtgacgc actggccgaa gtccgcatga ctaactcagg tgtccagctg gccggcggcg 25800 cttcccggtg cccgctccgc ccacaatcgg gtataaaaac cctggtgatc cgaggcagag 25860 gcacacagct caacgacgag ttggtgagct cttcgatcgg tctgcgaccg gacggagtgt 25920 tccaactagc cggagccggg agatcgtcct tcactcccaa ccaggcctac ctgaccttgc 25980 agagcagctc ttcggagcct cgctccggag gcatcggaac cctccagttc gtggaggagt 26040 ttgtgccctc ggtctacttc aaccccttct cgggatcgcc aggcctctac ccggacgagt 26100 ttataccgaa cttcgacgca gtgagagaag cggtggacgg ctacgactga atgtcccatg 26160 gtgactcggc tgagctcgct cggttgaggc atctggacca ctgccgccgc ctgcgctgct 26220 tcgcccggga gagctgcgga ctcatctact ttgagtttcc cgaggagcac cccaacggcc 26280 ctgcacacgg agtgcggatc accgtagagg gcaccaccga gtctcacctg gtcaggttct 26340 tcacccagca acccttcctg gtcgagcggg accggggagc taccacctac accgtctact 26400 gcatctgtcc taccccgaag ttgcatgaga atttttgctg tactctttgt ggtgagttta 26460 ataaaagctg aactaagaac cttctttgga atcccttgtc atcatcaaat caacaagacc 26520 atcaacttca cctttgagga acaggtgaac tttacctgca agccacacaa gaagtacatc 26580 atctggtttt atcacaacac tactctagca gtagccaaca cctgctcgaa cgacggtgtt 26640 ctcctaccta acaatctcac cagtggacta accttctcag ttaaaagggc aaagctaatt 26700 cttcatcgcc ctattgtaga aggaacttac cagtgtcaga gcggaccttg cttccacagt 26760 ttcactttgg tgaacgttac cggcagcagc acagccgctc cagaaacatc taaccttctt 26820 tctgatacta acaaacctcg tgtcggaggt gagctttggg ttccatctct aacagagggt 26880 gggagttcta ttgaagtggt tgggtatttg attttagggg tggtcattgg tgggtgcata 26940 gcagtgctgt atcaacttcc ttgctgggtc gaaatcaggg tatttatctg ctgggtcaga 27000 cattgtgggg aggaaccatg aaggggctct tgctgattat cctttccctg gtggggggtg 27060 tgctgtcatg ccacgaacag ccacgatgta acattaccac aggcaatgag aggaacgact 27120 gctctgtagt tatcaaatgc gagcaccatt gtcctctcaa catcacattc aagaataaga 27180 ccatgggaaa tgtatgggtg ggattctggc aaccaggaga tgagcagaac tacacggtca 27240 ctgtccatgg tagcgatggc aatcacactt tcggtttcaa attcattttt gaagtcatgt 27300 gtgatatcac actacatgtg gctagacttc atggcttgtg gccccctacc aaggagaaca 27360 tggtgggttt ttctttggct tttgtgatca tggcctgctt gatgtcaggt ctgctggtag 27420 gggctctagt gtggtttctg aaacgcaagc ccaggtacgg aaatgaggag aaggaaaaat 27480 tgctataaat tctttttctc ttcgcacaac catgaataca gtgttccgta tcgtgctgct 27540 ctctcttctt gtagctttcg gtcaggcagg aattcatatt attaatgcta catggtggga 27600 taatataact ttagtgggac cctcagatac tccagttacc tggtatgatg gcaagggatt 27660 gcaattttgt gacggaagta cagttaagaa tccgcagatc agacatactt gtaatgatca 27720 aaacttaact ctgattcatg ttaacaaaac ccatgaaaga acatacatgg gttacagaca 27780 tgacagtaag ggaaaagtag actataaggt tacagtcatt ccacctcctc ctgctactgt 27840 aaagccacaa ccagatccag aaaatgtctt tgtttatatg ggaaataatg taactttagt 27900 tggacctcca ggaattccag ttagttggta ttatcataat ggcacacagt tctgcgatgg 27960 agataaaatt attcatccag aattcaacca cacctgtgat aaacaaaacc ttacactgct 28020 gtttgtaaac tttacacatg atggaggcta tcttggattc aattacaaag gtactcagag 28080 aattcagtat gaggttatag ttttagatcg atttccaaat tctggtcaga tgaaaattga 28140 agaacaaagt gaggaaacag aacagaaaca tactgagcat aataaggctg gacaaaagca 28200 gggtatagat acaaatcaaa agaaagctaa taacagacaa aagccatctc aaaggccatc 28260 aagaagacgg ccgacaaaca ctcctgagac aaaacaactt acagtgtcta ttgggtctaa 28320 cttaacttta gttggtccag atggaaaagt cacttggtat gatggtgatt taaaaagacc 28380 atgtgaagaa caaaactata ggcttccaca tcagtgtagt gctcagaact taactttaat 28440 taatgtaact aaatctcatg agggaactta ctatggcact aatgacaaag acgaaagcaa 28500 aagatacaga gtgaaagtga acactacaaa ttctcaagct gtaaaaatta acccatatac 28560 cagacctact actcctgatc agaaacacag atttgaatta caaattgaaa ataatgcaaa 28620 tgatgaagaa tcaaaaattc catctactac tgtggcaatc gtggtgggag tgattgcggg 28680 cttcataact ataatcattg tcattctgtg ctacatctgc tgccgcaagc gtcccagggc 28740 atacaatcat atggtagacc cactactcag cttctcttac tgagactcag tcactttcat 28800 ttcagaacca tgaaggcttt cacagcttgc gttctgttta acataatcac acttagtgta 28860 gctgcaaatg gttttaaaca tgttaatgtt accagattaa gtaatgtaac actgacagga 28920 gctggaatta atactacatg gacagggtat tttaatgagg gtccaaaagg aaaaaatggg 28980 tggatgaata tttgcacatg gggcgatcct agatatgtgt gccatggaaa tagcagtact 29040 attactaatc ttacagttgt ggcacttcta aatttaacca ctaacagaag atttaaagca 29100 gaaagtttta ctagtaacga tggttatgaa actaccagtg caaaatttta tgaaattaaa 29160 attattgagc ttccaacaac tagagcaccc accacagtta ggacaacaca gcctaccact 29220 gtgcccacta cacatccaac caccacagtc agtacaacta ttgagaccac tactcatact 29280 acacagctag acacaacagt gcagaatact actttattga ttgggttttt actgagagga 29340 aatgaaagta ctactgaaca gacagaggct acctcaagtg ccttcagcag cactgcaaat 29400 ttaacttcgc ttgcttggac taatgaaacc ggagtatcat tgatgaatcg acagccttac 29460 tcaggtttgg atattcaaat tacttttctg gttgtctgtg ggatctttat tcttgcggtt 29520 cttctgtact ttgtctgctg caaagccaga gagaaatcta ggcggcccat atacaggcca 29580 gtaatcgggg aacctcagcc tctccaagtg gatggaggct taaggaatct tctcttctct 29640 tttacagtat ggtgatcagc catgattcct aggttcttcc tatttaacat cctgttctgt 29700 ctcttcaaca tctgtgctgc cttcgcggcc gtctcgcacg cctcgcccga ctgtctaggg 29760 cctttcccaa catacctcct ctttgccctg ctaacctgca cctgcgtctg cagcattgtc 29820 tgcgtggtca tcacctttct gcagctcatc gactggtgct gcgcgcgcta caattatctc 29880 caccacagtc ccgaatacag ggacgagaac gtagccagaa tcttaaggct catctgacca 29940 tgcagcctct gctcatgctg atatccctcc tatcccctgc ccttgccact tctgctgatt 30000 actctaaatg caaattcgcg gacatatgga atttcttaga ttgctatcag gagaaaattg 30060 atatgccctc ctattacttg gtgattgttg gggtagtcat ggtctgctca tgcactttct 30120 ttgccattat gatctacccc tgttttaatc ttggctggaa ctctgttgag gcattcacat 30180 acacactaga aaacagttca ctagcctcca cgccaccacc cacaccgcct ccccgcagaa 30240 atcagttccc tatgattcag tacttagaag agccccctcc ccggccccct tccactgtta 30300 gctactttca cataaccggc ggcgatgact gaccacctgg acctcgagat ggacggccag 30360 gcctccgagc agcgcatcct gcaactgcgc gtccgacagc agcaggagcg ggccgccaag 30420 gagctcctcg atgccatcaa catccaccag tgcaagaagg gcatcttctg cctggtcaag 30480 caggcaaaga tcacctacga gctcgtgtcc ggcggcaagc agcatcgcct cgcctatgag 30540 ctaccccagc agaagcaaaa gttcacctgc atggtgggcg tcaaccccat agtcatcacc 30600 cagcagtcgg gcgagaccaa cggctgcatc cactgctcct gcgaaagccc cgagtgcatc 30660 tactccctcc tcaagaccct ttgcggactc cgcgacctcc tccccatgaa ctgatgttga 30720 ttaaaagccc aaaaaccaat caaacccttc cccaattact cataagaata aatcattgga 30780 actaatcatt caataaagat cacttacttg aaatctgaaa gtatgtctct ggtgtagttg 30840 ttcagcagca cctcggaacc ctcctcccag ctctggtact ccagtccccg gcgggcggcg 30900 aacttcctcc acaccttgaa agggatgtca aattcctggt ccacaatttt cattgtcttc 30960 cctcagatga caaagaggct ccgggtggaa gatgacttca accccgtcta cccctatggc 31020 tacgcgcgga atcagaatat ccccttcctt actcccccct ttgtttcttc cgatggattc 31080 caaaacttcc cacctggggt cctgtcactc aaactggctg acccaatcgc catcactaat 31140 ggggatgttt cactcaaggt gggagggggt cttactgttg aaaaagatag tggaaatcta 31200 aaggtgaacc ctaaggctcc cttgcaagtt acaactgata aacagttgga aattgcactg 31260 gcttatccat ttgaagtcag taatggcaag cttggcataa aagcaggtca tggattgaaa 31320 gtcattgaca aaattgctgg tttggaaggt ttggcaggta cgcttgtagt tttgactgga 31380 aaaggaatag gtactgaaaa tcttgaaaac agtgatgggt caagtagagg agttggtata 31440 aacgtaagac ttgctaaaga tggaggtctg tcttttgata aaaagggtga tttagttgct 31500 tggaataaac atgatgacag acgcactcta tggacaactc ccgacccatc cccaaattgt 31560 acaatcgatc aggaaaggga ttcaaagctc actttagtat taacaaaatg tggcagtcaa 31620 attttggcta atgtctcttt acttgttgta aaaggaaaat ttagtaacat aaacaataat 31680 actaatccaa ctgataaaaa aatcacagta aagctacttt ttaatgaaaa gggagtatta 31740 atggacagtt cgacacttaa gaaagaatat tggaactaca gaaatgataa ttctactgta 31800 tctcaggcct atgataatgc agttcctttt atgccaaaca taaaagctta tcctaaacct 31860 accacagaca cttcggctaa accagaagat aaaaaaagtg ctgctaaaag atacattgtg 31920 agcaatgtct atattggagg cttgccagat aaaactgttg ttataactat taagtttaat 31980 gcagaaactg aatgtgctta ttcgattacc tttgaattca catgggcaaa aacctttgaa 32040 gatgtgcagt ttgattcctc ctcttttacc ttttcctata ttgcccaaga aaatgaggac 32100 gaagacaaat aaaatgtttt aaaatgaatt catgtatctt tattgatttt tacaccagca 32160 cgggtagtca gtctcccacc accagcccat ttcacagtgt aaacgattct ctcagcacgg 32220 gtggccttaa atagggaaat gttctgatta gtgcgggaac tggacttggg gtctataatc 32280 cacacagttt cctggcgagc caaacggggg tcggtgattg agatgaagcc gtcctctgaa 32340 aagtcatcca agcgggcctc acagtccaag gtcacagtct ggtgaaacga gaagaacgca 32400 cagattcata ctcggaaaac aggatgggtc tgtgcctctc catcagcgcc ctcaacagtc 32460 tctgccgccg gggctcggtg cggctgctgc agatgggatc gggatcacaa gtctctctga 32520 ctatgatccc cacagccttc agcatcagtc tcctggtgcg tcgggcacag caccgcatcc 32580 tgatctcgct catgttctca cagtaagtgc agcacataat caccatgtta ttcagcagcc 32640 cataattcag ggtgctccag ccaaaactca tgttggggat gatggaaccc acgtgaccat 32700 cgtaccagat gcggcagtat atcagatgcc tgcccctcat gaacacactg cccatataca 32760 tgatctcttt gggcatgtct ctgttcacaa tctgacggta ccagggaaag cgctggttga 32820 acatgcaccc gtaaatgact ctcctgaacc acacggccag cagggtgcct cccgcccgac 32880 actgcaggga gcccggggat gaacagtggc aatgcaggat ccagcgctcg tacccgctca 32940 ccatctgagc tctcaccaag tccagggtag cggggcacag gcacactgac atacatcttt 33000 ttaaaatttt tatttcctct ggagtcaaga tcatatccca ggggactgga aactcttgga 33060 gcagggtaaa gccagcagca catggtaatc cacggacaga acttacatta tgataatctg 33120 catgatcaca atcaggcaac aggggatgtt gttcagtcag tgaagccctg gtttcctcat 33180 cagatcgtgg taaacgggcc ctgcgatatg gatgatggcg gagcgagctg gattgaatct 33240 cggtttgcat tgtagtggat tctcttgcgt accttgtcgt acttctgcca gcagaaatgg 33300 gcccttgaac agcagatacc cctcctgcgg ccgtcctttc gctgctgccg ctcagtcatc 33360 caactgaagt acatccattc tcgaagattc tggagaagtt cctctgcatc tgatgaaaca 33420 aaaaacccgt ccatgcgaat tcccctcatc acatcagcca ggactctgta ggccatcccc 33480 atccagttaa tgctgccttg tctatcattc agagggggcg gtggcaggat tggaagaacc 33540 atttttattc caaacggtct cgaaggacga taaagtgcaa gtcacgcagg tgacagcgtt 33600 cccctccgct gtgctggtgg aaacagacag ccaggtcaaa acccactcta ttttcaaggt 33660 gctcgaccgt ggcttcgagc agtggctcta cgcgtacatc cagcataaga atcacattaa 33720 aggctggccc tccatcgatt tcatcaatca tcaggttaca ttcctgcacc atccccaggt 33780 aattctcatt tttccagcct tggattatct ctacaaattg ttggtgtaag tccactccgc 33840 acatgtggaa aagctcccac agtgccccct ccactttcat aatcaggcag accttcataa 33900 tagaaacaga tcctgctgct ccaccacctg cagcgtgttc aaaacaacaa gattcaataa 33960 ggttctgccc tccgccctga gctcgcgcct caatgtcagc tgcaaaaaat cacttaagtc 34020 ctgggccact acagctgaca attcagagcc agggctaagc gtgggactgg caagcgtaag 34080 ggaaaacttt aatgctccaa agctagcacc caaaaactgc atgctggaat aagctctctt 34140 tgtgtctccg gtgatgcctt ccaaaatgtg agtgataaag cgtggtagtt tttctttaat 34200 catttgcgta atagaaaagt cctgtaaata agtcactagg accccaggga ccacaatgtg 34260 gtagcttaca ccgcgtcgct gaagcatggt tagtagagat gagagtctga aaaacagaaa 34320 gcatgcacta aactaaggtg gctattttca ctgaaggaaa aatcactctc tccaacaaca 34380 gggtacccac tgggtggccc ttgcggacat acaaaaatcg gtccgtgtga ttaaaaagca 34440 gcacagtaag ttcctgtctt cttccggcaa aaatcacatc ggactgggtt agtatgtccc 34500 tggcatggta gtcattcaag gccataaatc tgccctgata tccagtagga accagcacac 34560 tcacttttag gtgaagcaat accaccccat gcggaggaat gtggaaagat tcagggcaaa 34620 aaaaattata tctattgcta gtcccttcct ggacgggagc aatccctcca ggactatcta 34680 tgaaagcata cagagattca gccatagctc agcccgctta ccagtagaca gagagcacag 34740 cagtacaagc gccaacagca gcgactgact acccactgac ccagctccct atttaaaggc 34800 gccttacact gacgtaatga ccaaaggtct aaaaaccccg ccaaaaaaaa acacacacgc 34860 cctgggtgtt ttttgcgaaa acacttccgc gttctcactt cctcgtattg atttcgtgac 34920 ttaacttccg ggttcccacg ttacgtcact tctgccctta catgtaactc agtcgtaggg 34980 cgccatcttg cccacgtcca aaatggcttc catgtccagc cacgcctccg cggcgaccgt 35040 tagccgtgcg tcgtgacgtc atttgcatca tcttctctcg tccaatcagc gctggccccg 35100 ccctaaattc aaaagctcat ttgcatgtta acttttgttt actttgtggg gtatattatt 35160 gatgatc 35167 6 19 DNA Artificial Sequence oligonucleotide 6 actcgagatg tatagatct 19 7 20 DNA Artificial Sequence oligonucleotide 7 ctagatctat acatctcgag 20 8 26 DNA Artificial Sequence PCR primer 8 gcacagatct ttgcttcagg aatatg 26 9 29 DNA Artificial Sequence PCR primer 9 gagaactcga ggcctacatg ggggtagag 29 10 26 DNA Artificial Sequence PCR primer 10 cgagacggcc gacgcagatc tgtttg 26 11 25 DNA Artificial Sequence PCR primer 11 gaagtcccgg gctacatggg ggtag 25 12 34775 DNA adenovirus serotype 34 12 catcatcaat aatatacctt atagatggaa tggtgccaat atgtaaatga ggtgatttta 60 aaaattgtgg ggtgtgtggt gattggctgt ggggttaacg gctaaacggg gcggcgcggc 120 cgtgggaaaa tgacgttttg tgggggtgga gtttttttgc aagttgtcgc gggaaatgtg 180 acgcataaaa aggctttttt tctcacggaa ctactgactt ttcccacggt atttaacagg 240 aaatgaggta gttttgaccg gatgcaagtg aaaattgctg atttgcgcgc gaaaactgaa 300 tgaggaagtg tttttctgaa taatgtggta tttatggcag ggtggagtat ttgttcaggg 360 ccaggtagac tttgacccat tacgtggagg tttcgattac cgtgtttttt acctgaattt 420 ccgcgtaccg tgtcaaagtc ttctgttttt acgtaggtgt cagctgatcg ctacggtatt 480 tatacctcag ggtttgtgtc aagaggccac tcttgagtgc cagcgagaag agttttctcc 540 tctgcgccgg cagtttaata ataaaaaaat gagagatttg cgatttctgc ctcaggaaat 600 aatttctgct gagactggaa atgaaatact ggagcttgtg gtgcacgccc tgatgggaga 660 cgatccggag ccacctgtgc agctttttga gcctcctacg cttcaggaac tgtatgattt 720 agaggtagag ggatcggagg attctaatga ggaagctgtg aatggctttt ttaccgattc 780 tatgctttta gctgctaatg aaggattaga attagatccg cctttggaca ctttcgatac 840 tccaggggtg attgtggaaa gcggtacagg tgtaagaaaa ttacctgatt tgggttccgt 900 ggactgtgat ttgcactgct atgaagacgg gtttcctccg agtgatgagg aggaccatga 960 aaaggagcag tctatgcaga ctgcagcggg tgagggagtg aaggctgcca gtgttggttt 1020 tcagttggat tgcccggagc ttcctggaca tggctgtaag tcttgtgaat ttcacaggaa 1080 aaatactgga gtaaaggaac tgttatgttc gctttgttat atgagagcgc actgccactt 1140 tatttacagt aagtgtgttt aagttaaaat ttaaaggaat atgctgtttt tcacatgtat 1200 attgagtggg agttttgtgc ttcttattat aggtcctgtg tctgatgctg atgagtcacc 1260 atctcctgat tctactacct cacctcctga gattcaagca cctgttcctg tggacgtgcg 1320 caagcccatt cctgtgaagc ttaagcctgg gaaacgtcca gcagtggaaa aacttgagga 1380 cttgttacag ggtggggacg gacctttgga cttgagtaca cggaaacggc caagacaata 1440 agtgttccat atccgtgttt acttaaggtg acgtcaatat ttgtgtgaga gtgcaatgta 1500 ataaaaatat gttaactgtt cactggtttt tattgctttt tgggcgggga ctcaggtata 1560 taagtagaag cagacctgta tggttagctc ataggagctg gctttcatcc atggaggttt 1620 gggccatttt ggaagacctt agaaagacta ggcaactgtt agaggacgct tcggacggag 1680 tctccggttt ttggagattc tggttcgcta gtgaattagc tagggtagtt tttaggataa 1740 aacaggacta taaagaagaa tttgaaaagt tgttggtaga ttgcccagga ctttttgaag 1800 ctcttaattt gggccatcaa gttcacttta aagaaaaagt tttatcagtt ttagactttt 1860 caaccccagg tagaactgcc gctgctgtgg cttttcttac ttttatatta gataaatgga 1920 tcccgcagac tcatttcagc aggggatacg ttttggattt cgtagccaca gcattgtgga 1980 gaacatggaa ggttcgcaag atgaggacaa tcttaggtta ctggccagtg cagcctttgg 2040 gtgtagcggg aatcctgagg catccaccgg tcatgccagc ggttctggag gaggaacagc 2100 aagaggacaa cccgagagcc ggcctggacc ctccagtgga ggaggcggag tagctgactt 2160 gtctcctgaa ctgcaacggg tgcttactgg atctacgtcc actggacggg ataggggcgt 2220 taagagggag agggcatcta gtggtactga tgctagatct gagttggctt taagtttaat 2280 gagtcgcaga cgtcctgaaa ccatttggtg gcatgaggtc cagaaagagg gaagggatga 2340 agtttctgta ttgcaggaga aatattcact ggaacaggtg aaaacatgtt ggttggagcc 2400 tgaggatgat tgggaggtgg ccattaaaaa ttatgccaag atagctttga ggcctgataa 2460 acagtataag attactagac ggattaatat ccggaatgct tgttacatat ctggaaatgg 2520 ggctgaggtg gtaatagata ctcaagacaa ggcagttatt agatgctgca tgatggatat 2580 gtggcctgga gtagtcggta tggaagcagt aacttttgta aatgttaagt ttaggggaga 2640 tggttataat ggaatagtgt ttatggccaa taccaaactt atattgcatg gttgtagctt 2700 ttttggtttc aacaatacct gtgtagatgc ctggggacag gttagtgtac ggggatgtag 2760 tttctatgcg tgttggattg ccacagctgg cagaaccaag agtcaattgt ctctgaagaa 2820 atgcatattc caaagatgta acctgggcat tctgaatgaa ggcgaagcaa gggtccgcca 2880 ctgcgcttct acagatactg gatgttttat tttaattaag ggcaatgcca gcgtaaagca 2940 taacatgatt tgcggtgctt ccgatgagag gccttatcaa atgctcactt gtgccggtgg 3000 gcattgtaat atgctggcta ctgtgcatat tgtttcccat caacgcaaaa aatggcctgt 3060 ttttgatcac aatgtgttga ccaagtgtac catgcatgca ggtgggcgta gaggaatgtt 3120 tatgccttac cagtgtaaca tgaatcatgt gaaagtgttg ttggaaccag atgccttttc 3180 cagaatgagc ctaacaggaa tctttgacat gaacatgcaa atctggaaga tcctgaggta 3240 tgatgatacg agatcgaggg tgcgcgcatg cgaatgcgga ggcaagcatg ccaggttcca 3300 gccggtgtgt gtagatgtga ctgaagatct gagaccggat catttggtta ttgcccgcac 3360 tggagcagag ttcggatcca gtggagaaga aactgactaa ggtgagtatt gggaaaactt 3420 ggggtggggt tttcagatgg acagattgag taaaaatttg ttttttctgt ctttcagctg 3480 tcatgagtgg aaacgcttct tttaaggggg gagtcttcag cccttatctg acagggcgtc 3540 tcccatcctg ggcaggagtt cgtcagaatg ttatgggatc tactgtggat ggaagacccg 3600 tccaacccgc caattcttca acgctgacct atgctacttt aagttcttca cctttggacg 3660 cagctgcagc cgccgccgcc gcctctgttg ccgctaacac tgtgcttgga atgggttact 3720 atggaagtat cgtggctaat tccacttcct ctaataaccc ttctaccctg actcaggaca 3780 agttacttgt ccttttggcc cagctggagg ctttgaccca acgtctgggt gaactttatc 3840 agcaggtggc cgagttgcga gtacaaactg agtctgctgt cggcacggca aagtctaaat 3900 aaaaaaaaat tccacaatca atgaataaat aaacgagctt gttgttgatt taaaatcaag 3960 tgtttttatt tcatttttcg cgcacggtat gccctagacc accgatctcg atcattgaga 4020 acacggtgga ttttttccag aatcctatag aggtgggatt gaatgtttag atacatgggc 4080 attaggccat ctttggggtg gagatagctc cattgaaggg attcatgctc cggggtagtg 4140 ttgtaaatca cccagtcata acaaggtcgc agtgcatggt gttgcacaat atcttttaga 4200 agtaggctga ttgccacaga taagcccttg gtgtaggtgt ttacaaaccg gttgagctgg 4260 gaggggtgca ttcggggtga aattatgtgc attttggatt ggatttttaa gttggcaata 4320 ttgccgccaa gatctcgtct tgggttcatg ttatgaagga ccaccaagac ggtgtatccg 4380 gtacatttag gaaatttatc gtgtagcttg gatggaaaag cgtggaaaaa tttggagaca 4440 cccttgtgtc ctccgagatt ttccatgcac tcatccatga taatagcaat ggggccgtgg 4500 gcagcagcgc gggcaaacac gttccgtggg tctgacacat catagttatg ttcctgagtt 4560 aaatcatcat aagccatttt aatgaatttg gggcggagag tacccgattg gggtatgaat 4620 gttccttcgg gccccggagc atagttcccc tcacagattt gcatttccca agctttcagt 4680 tccgatggtg gaatcatgtc cacctggggg gctatgaaga acaccgtttc tggggcgggg 4740 gtgattagtt gggatgatag caagtttctg agcaattgag atttgccaca tccggtgggg 4800 ccataaatga ttccgattac aggttgcagg tggtagttta gggaacggca actgccgtct 4860 tctcgaagca agggggccac ctcgttcatc atttccctta catgcatatt ttcccgcacc 4920 aaatccatta ggaggcgctc tcctcctagt gatagaagtt cttgtagtga ggaaaagttt 4980 ttcagcggtt ttagaccgtc agccatgggc attttggaga gagtttgctg caaaagttct 5040 agtctgttcc acagttcagt gatgtgttct atggcatctc gatccagcag acctcctcgt 5100 ttcgcgggtt tggacggctc ctggagtagg gtatgagacg atgggcgtcc agcgctgcca 5160 gggttcggtc cttccagggt ctcagtgttc gagtcagggt tgtttccgtc acagtgaagg 5220 ggtgtgcgcc tgcttgggcg cttgccaggg tgcgcttcag actcattctg ctggtggaga 5280 acttctgtcg cttggcgccc tgtatgtcgg ccaagtagca gtttaccatg agttcgtagt 5340 tgagcgcctc ggctgcgtgg cctttggcgc ggagcttacc tttggaagtt ttcttgcata 5400 ccgggcagta taggcatttc agcgcataca gcttgggcgc aaggaaaatg gattctgggg 5460 agtatgcatc tgcgccgcag gaggcgcaaa cagtttcaca ttccaccagc caggttaaat 5520 ccggttcatt ggggtcaaaa acaagttttc cgccatattt tttgatgcgt ttcttacctt 5580 tggtctccat gagttcgtgt cctcgttgag tgacaaacag gctgtccgta tccccgtaga 5640 ctgattttac aggcctcttc tccagtggag tgcctcggtc ttcttcgtac aggaactctg 5700 accactctga tacaaaggcg cgcgtccagg ccagcacaaa ggaggctatg tgggaggggt 5760 agcgatcgtt gtcaaccagg gggtccacct tttccaaagt atgcaaacac atgtcaccct 5820 cttcaacatc caggaatgtg attggcttgt aggtgtattt cacgtgacct ggggtccccg 5880 ctgggggggt ataaaagggg gcggttcttt gctcttcctc actgtcttcc ggatcgctgt 5940 ccaggaacgt cagctgttgg ggtaggtatt ccctctcgaa ggcgggcatg acctctgcac 6000 tcaggttgtc agtttctaag aacgaggagg atttgatatt gacagtgccg gttgagatgc 6060 ctttcatgag gttttcgtcc atttggtcag aaaacacaat ttttttattg tcaagtttgg 6120 tggcaaatga tccatacagg gcgttggata aaagtttggc aatggatcgc atggtttggt 6180 tcttttcctt gtccgcgcgc tctttggcag cgatgttgag ttggacatac tcgcgtgcta 6240 ggcacttcca ttcggggaag atagttgtca attcatctgg cacgattctc acttgccacc 6300 ctcgattatg caaggtaatt aaatccacac tggtggccac ctcgcctcga aggggttcgt 6360 tggtccaaca gagcctacct cctttcctag aacagaaagg gggaagtggg tctagcataa 6420 gttcatcggg agggtctgca tccatggtaa agattcccgg aagtaaatcc ttatcaaaat 6480 agctgatggg agtggggtca tctaaggcca tttgccattc tcgagctgcc agtgcacgct 6540 catatgggtt aaggggactg ccccagggca tgggatgggt gagtgcagag gcatacatgc 6600 cacagatgtc atagacgtag atgggatcct caaagatgcc tatataggtt ggatagcatc 6660 gcccccctct gatacttgct cgcacatagt catatagttc atgtgatggc gctagcaacc 6720 ccggacccaa gttggtgcga ttgggttttt ctgttctgta gacaatctgg cgaaagatgg 6780 cgtgagaatt ggaagagatg gtgggtcttt gaaaaatgtt gaaatgggca tgaggtagac 6840 ctacagagtc tctgacaaag tgggcataag attcttgaag cttggttacc agttcggcgg 6900 tgacaagtac gtctagggcg cagtagtcaa gtgtttcttg aatgatgtca taacctggtt 6960 ggtttttctt ttcccacagt tcgcggttga gaaggtattc ttcgcgatcc ttccagtact 7020 cttctagcgg aaacccgtct ttgtctgcac ggtaagatcc tagcatgtag aactgattaa 7080 ctgccttgta agggcagcag cccttctcta cgggtagaga gtatgcttga gcagcttttc 7140 gcagcgaagc gtgagtaagg gcgaaggtgt ctctgaccat gactttgaga aattggtatt 7200 tgaagtccat gtcgtcacag gctccctgtt cccagagttg gaagtctacc cgtttcttgt 7260 aggcggggtt gggcaaagcg aaagtaacat cgttgaagag aatcttaccg gctctgggca 7320 taaaattgcg agtgatgcgg aaaggctgtg gtacttccgc tcgattgttg atcacctggg 7380 cagctaggac gatctcgtcg aaaccgttga tgttgtgtcc tacgatgtat aattctatga 7440 aacgcggcgt gcctttgacg tgaggtagct tattgagctc atcaaaggtt aggtctgtag 7500 ggtcagataa ggcgtagtgt tcgagagccc attcgtgcag gtgaggattt gcatgtagga 7560 atgatgacca aagatccacc gccagtgctg tttgtaactg gtcccgatac tgacgaaaat 7620 gctggccaat tgccattttt tctggagtga cacagtagaa ggttctgggg tcttgttgcc 7680 atcgatccca ctttagttta atggctagat cgtgggccat gttgacgaga cgctcttctc 7740 ctgagagttt catgaccagc atgaaaggaa ctagttgttt gccaaaggac cccatccagg 7800 tgtaagtttc cacatcgtag gtcaggaaga gtctttctgt gcgaggatga gagccgatcg 7860 ggaagaactg gatttcctgc caccagttgg aggattggct gttgatgtga tggaagtaga 7920 agtttctgcg gcgcgccgag cattcgtgtt tgtgcttgta cagacggccg cagtagtcgc 7980 agcgttgcac gggttgtatc tcgtgaatga gctgtacctg gcttcccttg acgagaaatt 8040 tcagtgggaa gccgaggcct ggcgattgta tctcgtgctc ttctatattc gctgtatcgg 8100 cctgttcatc ttctgtttcg gtggtggtca tgctgacgag cccccgcggg aggcaagtcc 8160 agacctcggc gcgggagggg cggagctgaa ggaccagagc gcgcaggctg gagctgtcca 8220 gagtcctgag acgctgcgga ctcaggttag taggtaggga cagaagatta acttgcatga 8280 tcttttccag ggcgtgcggg aggttcagat ggtacttgat ttccacaggt tcgtttgtag 8340 agatgtcaat ggcttgcagg gttccgtgtc ctttgggcgc cactaccgta cctttgtttt 8400 ttcttttgat cggtggtggc tctcttgctt cttgcatgct cagaagcgat gacggggacg 8460 cgcgccgggc ggaagcggtt gttccggacc cggaggcatg gctggtagtg gcacgtcggc 8520 gccgcgcacg ggcaggttct ggtactgcgc tctgagaaga cttgcgtgcg ccaccacgcg 8580 tcgattgacg tcttgtatct gacgtctctg ggtgaaagct accggccccg tgagcttgaa 8640 cctgaaagag agttcaacag aatcaatttc ggtatcgtta acggcagctt gtctcagtat 8700 ttcttgtacg tcaccagagt tgtcctggta ggcgatctcc gccatgaact gctcgatttc 8760 ttcctcctga agatctccgc gacccgctct ctcgacggtg gccgcgaggt cattggagat 8820 acggcccatg agttgggaga atgcagtcat gcccgcctcg ttccagacgc ggctgtaaac 8880 cacggccccc tcggagtctc ttgcgcgcat caccacctga gcgaggttaa gctccacgtg 8940 tctggtgaag accgcatagt tgcataggcg ctgaaaaagg tagttgagtg tggtggcaat 9000 gtgttcggcg acgaagaaat acatgatcca tcgtctcagc ggcatttcgc tgacatcgcc 9060 cagagcttcc aagcgctcca tggcctcgta gaagtccacg gcaaaattaa aaaactggga 9120 gtttcgcgcg gacacggtca attcctcctc gagaagacgg atgagttcgg ctatggtggc 9180 ccgtacttcg cgttcgaagg ctcccgggat ctcttcttcc tcttctatct cttcttccac 9240 taacatctct tcttcgtctt caggcggggg cggagggggc acacggcgac gtcgacggcg 9300 cacgggcaaa cggtcgatga atcgttcaat gacctctccg cggcggcggc gcatggtttc 9360 agtgacggcg cggccgttct cgcgcggtcg cagagtaaaa acaccgccgc gcatctcctt 9420 aaagtggtga ctgggaggtt ctccgtttgg gagggagagg gcgctgatta tacattttat 9480 taattggccc gtagggactg cgcgcagaga tctgatcgtg tcaagatcca cgggatctga 9540 aaacctttcg acgaaagcgt ctaaccagtc acagtcacaa ggtaggctga gtacggcttc 9600 ttgtgggcgg gggtggttat gtgttcggtc tgggtcttct gtttcttctt catctcggga 9660 aggtgagacg atgctgctgg tgatgaaatt aaagtaggca gttctaagac ggcggatggt 9720 ggcgaggagc accaggtctt tgggtccggc ttgctggata cgcaggcgat tggccattcc 9780 ccaagcatta tcctgacatc tagcaagatc tttgtagtag tcttgcatga gccgttctac 9840 gggcacttct tcctcacccg ttctgccatg catacgtgtg agtccaaacc cgcgcattgg 9900 ttgtaccagt gccaagtcag ctacgactct ttcggcgagg atggcttgct gtacttgggt 9960 gagggtggct tgaaagtcat caaaatccac aaagcggtgg taagccccgg tattaatggt 10020 gtaagcacag ttggccatga ctgaccagtt aactgtctgg tgaccagggc gcacgagctc 10080 ggtgtattta aggcgcgaat aggcgcgggt gtcaaagatg taatcgttgc aggtgcgcac 10140 cagatactgg taacctataa gaaaatgcgg cggtggttgg cggtagagag gccatcgttc 10200 tgtagctgga gcgccggggg cgaggtcttc caacataagg cggtgatagc cgtagatgta 10260 cctggacatc caggtgattc ctgcggcggt agtagaagcc cgaggaaact cgcgtacgcg 10320 gttccaaatg ttgcgtagcg gcatgaagta gttcattgta ggcacggttt gaccagtgag 10380 gcgcgcgcag tcattgatgc tctatagaca cggagaaaat gaaagcgttc agcgactcga 10440 ctccgtagcc tggaggaacg tgaacgggtt gggtcgcggt gtaccccggt tcgagacttg 10500 tactcgagcc ggccggagcc gcggctaacg tggtattggc actcccgtct cgacccagcc 10560 tacaaaaatc caggatacgg aatcgagtcg ttttgctggt tgccgaatgg cagggaagtg 10620 agtcctattt tttttttttg ccgctcagat gcatcccgtg ctgcgacaga tgcgtcccca 10680 acaacagccc ccctcgcagc agcagcaacc acaaaaggct gtccctgcaa ctactgcaac 10740 tgccgctgtg agcggtgcgg gacagcccgc ctatgatctg gacttggaag agggcgaagg 10800 actggcacgt ctaggtgcgc cttcgcccga gcggcatccg cgagttcaac tgaaaaaaga 10860 ttctcgcgag gcgtatgtgc cccaacagaa cctatttaga gacagaagcg gcgaggagcc 10920 ggaggagatg cgagcttccc gctttaacgc gggtcgtgag ctgcgtcacg gtttggacag 10980 aagacgagtg ttgcgggacg aggatttcga agttgatgaa gtgacaggga tcagtcctgc 11040 cagggcacac gtggctgcag ccaaccttgt atcggcttac gaacagacag taaaggaaga 11100 gcgtaatttc caaaagtctt ttaataatca tgtgcgaacc ctcattgccc gcgaagaagt 11160 cacccttggt ttgatgcatt tgtgggattt gatggaagct atcattcaga accctactag 11220 caaacctctg accgcacagc tgtttctggt ggtgcaacac agcagagaca atgaggcttt 11280 cagagaggcg ctgctcaaca tcaccgaacc cgaggggaga tggttgtatg atcttatcaa 11340 cattctacag agtatcatag tgcaggagcg gagcctgggc ctggccgaga aggtggctgc 11400 catcaattac tcggttttga gcttgggaaa gtattacgct cgcaagatct acaagactcc 11460 atacgttccc atagacaagg aggtgaagat agatgggttc tacatgcgca tgacgctgaa 11520 ggtgttgacc ctgagcgatg atcttggggt gtaccgcaat gacagaatgc atcgcgcggt 11580 gagcgccagc aggaggcgcg agttaagcga cagggaactg atgcacagtt tgcaaagagc 11640 tctaactgga gctggaaccg agggtgagaa ttactttgat atgggagctg acttgcagtg 11700 gcagcctagt cgcagggctc tgaacgccgc gacggcagga tgtgagcttc cttacataga 11760 agaggcggat gaaggcgagg aggaagaggg cgagtacttg gaagactgat ggcacaaccc 11820 gtgttttttg ctagatggaa cagcaagcac cggatcccgc aatgcgggcg gcgctgcaga 11880 gccagccgtc cggcattaac tcctcggacg attggaccca ggccatgcaa cgtatcatgg 11940 cgttgacgac tcgcaacccc gaagccttta gacagcaacc ccaggccaac cgtctatcgg 12000 ccatcatgga agctgtagtg ccttcccgct ctaatcccac tcatgagaag gtcctggcca 12060 tcgtgaacgc gttggtggag aacaaagcta ttcgtccaga tgaggccgga ctggtataca 12120 acgctctctt agaacgcgtg gctcgctaca acagtagcaa tgtgcaaacc aatttggacc 12180 gtatgataac agatgtacgc gaagccgtgt ctcagcgcga aaggttccag cgcgatgcca 12240 acctgggttc gctggtggcg ttaaatgctt tcttgagtac tcagcctgct aatgtgccgc 12300 gtggtcaaca ggattatact aactttttaa gtgctttgag actgatggta tcagaagtac 12360 ctcagagcga agtatatcag tccggtcctg attacttctt tcagactagc agacagggct 12420 tgcagacggt aaatctgagc caagctttta aaaaccttaa aggtttgtgg ggagtgcatg 12480 ccccggtagg agaaagagca accgtgtcta gcttgttaac tccgaactcc cgcctattat 12540 tactgttggt agctcctttc accgacagcg gtagcatcga ccgtaattcc tatttgggtt 12600 acctactaaa cctgtatcgc gaagccatag ggcaaagtca ggtggacgag cagacctatc 12660 aagaaattac ccaagtcagt cgcgctttgg gacaggaaga cactggcagt ttggaagcca 12720 ctctgaactt cttgcttacc aatcggtctc aaaagatccc tcctcaatat gctcttactg 12780 cggaggagga gaggatcctt agatatgtgc agcagagcgt gggattgttt ctgatgcaag 12840 agggggcaac tccgactgca gcactggaca tgacagcgcg aaatatggag cccagcatgt 12900 atgccagtaa ccgacctttc attaacaaac tgctggacta cttgcacaga gctgccgcta 12960 tgaactctga ttatttcacc aatgccatct taaacccgca ctggctgccc ccacctggtt 13020 tctacacggg cgaatatgac atgcccgacc ctaatgacgg atttctgtgg gacgacgtgg 13080 acagcgatgt tttttcacct ctttctgatc atcgcacgtg gaaaaaggaa ggcggcgata 13140 gaatgcattc ttctgcatcg ctgtccgggg tcattggtgc taccgcggct gagcccgagt 13200 ctgcaagtcc ttttcctagt ctaccctttt ctctacacag tgtacgtagc agcgaagtgg 13260 gtagaataag tcgcccgagt ttaatgggcg aagaggagta cctaaacgat tccttgctca 13320 gaccggcaag agaaaaaaat ttcccaaaca atggaataga aagtttggtg gataaaatga 13380 gtagatggaa gacttatgct caggatcaca gagacgagcc tgggatcatg gggactacaa 13440 gtagagcgag ccgtagacgc cagcgccatg acagacagag gggtcttgtg tgggacgatg 13500 aggattcggc cgatgatagc agcgtattgg acttgggtgg gagaggaagg ggcaacccgt 13560 ttgctcattt gcgccctcgc ttgggtggta tgttgtaaaa aaaaataaaa aagaaaaaac 13620 tcaccaaggc catggcgacg agcgtacgtt cgttcttctt tattatctgt gtctagtata 13680 atgaggcgag tcgtgctagg cggagcggtg gtgtatccgg agggtcctcc tccttcgtac 13740 gagagcgtga tgcagcagca gcaggcgacg gcggtgatgc aatccccact ggaggctccc 13800 tttgtgcctc cgcgatacct ggcacctacg gagggcagaa acagcattcg ttactcggaa 13860 ctggcacctc agtacgatac caccaggttg tatctggtgg acaacaagtc ggcggacatt 13920 gcttctctga actatcagaa tgaccacagc aacttcttga ccacggtggt gcaaaacaat 13980 gactttaccc ctacggaagc cagcacccag accattaact ttgatgaacg atcgcggtgg 14040 ggcggtcagc taaaaaccat catgcatact aacatgccca acgtgaacga gtatatgttt 14100 agtaacaagt tcaaagcgcg tgtgatggtg tccagaaaac ctcctgaggg tgttagagta 14160 gacgataatt atgatcataa gcaagatatt ctaaaatacg agtggttcga gtttactttg 14220 ccagaaggca acttttcggt cactatgact atcgacttga tgaacaatgc catcatagac 14280 aattacttga aagtgggcag acagaatgga gtgttggaaa gtgacattgg tgttaagttc 14340 gacactagga acttcaagtt gggatgggat ccagaaacta agttgatcat gcctggggtt 14400 tacacctatg aggccttcca tcctgacatc gtattgctgc ctggctgcgg agtggacttt 14460 accgaaagcc gtctgagcaa ccttcttggc attagaaaga aacacccatt ccaagagggt 14520 tttaagatct tgtatgagga tttagaagga ggaaatattc cagccctttt ggatgtagat 14580 gcttatgaga acagcaagaa agatcaaaaa gccaaaatag aagctgctgc agaagctaaa 14640 gcaaacatag ttgccaacga tccggtaagg gtggctaacg ctagtgaaat caggggagac 14700 agttttgccg caacatccgt tccgactaaa gaatcattat tggatgatgt gtctcaaaac 14760 atagagttaa aactcactat taagcctgtg gaaaaagatg gcaaaaacag aagttacaat 14820 gtgttggaag ataaaatcaa cacggcctat cgcagttggt acctttcgta caattatggc 14880 gaccccgaaa aaggagtgcg ttcctggaca ttgctcacca cctcagatgt cacctgcgga 14940 gcggagcagg tctactggtc gcttccagac atgatgcagg atcctgtcac tttccgctcc 15000 actagacaag tcagtaacta ccctgtggtg ggtgcagagc ttatgcccgt cttttcaaag 15060 agcttctaca acgaacaagc tgtgtactcc cagcagctcc gccagtccac ctcgcttacg 15120 cacgtcttca accgctttcc tgagaaccag attttaatcc gtccgccggc gcccacaatt 15180 accaccgtca gtgaaaacgt tcctgctctc acagatcacg ggaccctgcc gttgcgcagc 15240 agtatccggg gagtccaacg tgtgaccgtt actgacgcca gacgccgcac ctgtccctac 15300 gtgtacaagg cactgggcat agtcgcaccg cgcgtccttt caagccgcac tttctaaaaa 15360 aaaaaaaaaa atgtccgttc ttatctcgcc cagtaataac accggttggg gtctgcgcgc 15420 tcccagcaag atgtacggag gcgcacgcaa acgttctacc caacatcccg tgcgtgttcg 15480 cgggcatttt cgcgctccat ggggtgccct caagggccgc actcgcgttc gaaccaccgt 15540 cgatgatgta atcgatcagg tggttgccga cgcccgtaat tatactccta ctgcgcctac 15600 atctactgtg gacgcagtta ttgacagtgt agtggctgac gctcgcaact atgctcgacg 15660 taagagccgg cgaaggcgca ttgccagacg tcaccgagct accactgcca tgcgagcagc 15720 aagagctctg ctacgaagag ctagacgcgt ggggcgaaga gccatgctta gggcggccag 15780 acgtgcagct tcgggcgcca gcgccggcag gtcccgcagg caagcagccg ctgtcgcagc 15840 ggcgactatt gccgacatgg cccaatcgcg aagaggcaat gtatactggg tgcgtgacgc 15900 tgccaccggt caacgtgtac ccgtgcgcac ccgtccccct cgcacttaga agatactgag 15960 cagtctccga tgttgtgtcc cagcggcgag gatgtccaag cgcaaataca aggaagaaat 16020 gctgcaggtt atcgcacctg aagtctacgg ccaaccgttg aaggatgaaa aaaaaccccg 16080 caaaatcaag cgggtaaaaa aggacaaaaa agaagaggaa gatggcgatg atgggctggc 16140 ggagtttgtg cgcgagtttg ccccacggcg acgcgtgcaa tggcgtgggc gcaaagttcg 16200 acatgtgttg agacctggaa cttcggtggt ctttacaccc ggcgagcgtt caagcgctac 16260 ttttaagcgt tcctatgatg aggtgtacgg ggatgatgat attcttgagc aggcagctga 16320 ccgattaggc gagtttgctt atggcaagcg tagtagaata aatcccaagg atgaaacagt 16380 gtccataccc ttggatcatg gaaatcccac ccctagtctt aaaccggtca ctttgcagca 16440 agtgttaccc gtaactccgc gaacaggtgt taaacgcgaa ggtgaagatt tgtatcccac 16500 tatgcaactg atggtgccca aacgccagaa gttggaggac gttttggaga aagtaaaagt 16560 ggatccagat attcaacctg aggttaaagt gagacccatt aagcaggtag cgcctggtct 16620 gggagtacaa actgtagaca ttaaaattcc cactgaaagt atggaagtgc aaactgaacc 16680 cgcaaagcct actgccacct ccactgaagt gcaaacggac ccatggatgc ccatgcctat 16740 tacaactgac gccgtcggtc ccactcgaag atcccgacga aagtacggtc cagcaagtct 16800 gttgatgccc aactatgtcg tacacccatc tattattcct actcctggtt accgaggcac 16860 tcgctactat cgcagccgaa acagtacttc ccgccgtcgc cgcaagacac ctgcaaatcg 16920 cagtcgtcgc cgtagacgca caagcaaacc gattcccggc gccctggtgc ggcaagtgta 16980 ccgcaatggt agtgcggaac ctttgacact gccgcgtgcg cgttaccatc ctagtatcat 17040 cacttaatca atgttgccgc tgcctccttg cagatatggc cctcacttgt cgccttcgcg 17100 ttcccatcac tggttaccga ggaagaaact cgcgccgtag aagagggatg ttggggcgcg 17160 gaatgcgacg ctacaggcga cggcgtgcta tccgcaagca attgcggggt ggttttttgc 17220 cagccttaat tccaattatc gctgctgcga ttggcgcaat accaggcata gcttccgtgg 17280 cggttcaggc ctcgcaacga cattgacatt ggaaaaaaaa aaaacgtata aataaaaaat 17340 acaatggact ctgacactcc tggtactgtg actatgtttt cttagagatg gaagacatca 17400 atttttcatc cttggctccg cgacacggca cgaagccgta catgggcacc tggagcgaca 17460 tcggcacgag ccaactgaac gggggcgcct tcaattggag cagtatctgg agcgggctta 17520 aaaattttgg ctcaaccata aaaacatacg ggaacaaagc ttggaacagc agtacaggac 17580 aggcgcttag aaataaactt aaagaccaga acttccaaca aaaagtagtc gatgggatag 17640 cttccggtat caatggagtg gtagatttgg ctaaccaggc tgtgcagaaa aagataaaca 17700 gtcgtttgga cccgccgcca gcaaccccag gtgaaatgca agtggaggaa gaaattcctc 17760 cgccagaaaa acgaggcgac aagcgtccgc gtcccgattt ggaagagacg ctggtgacgc 17820 gcgtagatga accgccttct tatgaggaag caacgaagct tggaatgccc accactagac 17880 cgatagcccc tatggccacc ggggtgatga aaccttctca gttgcatcga cccgtcacct 17940 tggatttgcc ccctcctcct gctgctactg ctgtacccgc ttctaagcct gtcgctgccc 18000 cgaaaccagt cgccgtagcc aggtcacgtc ccgggggcgc tcctcgtcca aatgcacact 18060 ggcaaaatac tctgaacagc atcgtgggtc taggcgtgca aagtgtaaaa cgccgtcgct 18120 gcttttaatt aaatatggag tagcgcttaa cttgcctatc tgtgtatatg tgtcattaca 18180 cgccgtcaca gcatcagagg aaaaaaggaa gaggtcgtgc gtcgacgctg agttactttc 18240 aagatggcca ccccatcgat gctgccccaa tgggcataca tgcacatcgc cggacaggat 18300 gcttcggagt acctgagtcc gggtctggtg cagttcgccc gcgccacaga cacctacttc 18360 aatctgggaa ataagtttag aaatcctacc gtagcgccga cccacgatgt gaccaccgat 18420 cgtagccagc ggctcatgtt gcgcttcgtg cccgttgacc gggaggacaa tacatactct 18480 tacaaagtgc ggtacaccct ggccgtgggc gacaacagag tgctggatat ggccagcacg 18540 ttctttgaca ttaggggcgt gttggacaga ggtcccagtt ttaaacccta ttctggtacg 18600 gcttacaact ccctggctcc taaaggcgct ccaaatgcat ctcagtggtt ggataaggga 18660 gttacaagca ctggcctagt ggacgacggc aatactgatg atggggaaga agccaaaaaa 18720 gcaacataca cttttggtaa tgctccagta aaagccgagg ctgaaatcac aaaagacgga 18780 ttgccggtgg gcttggaagt ttcaactgaa ggtcctaaac caatctatgc tgataagctt 18840 tatcagccag aacctcaagt gggagacgaa acttggactg acctagacgg aaaaaccgaa 18900 gagtatggag ggagggttct taaacctgaa actaaaatga aaccctgcta cggatctttt 18960 gctaaaccta ctaatattaa aggaggtcag gcaaaggtaa aaccaaaaga agacgatggc 19020 actaacaaca tcgagtatga cattgacatg aacttctttg acttaagatc acaaagatca 19080 gaactcaaac ctaaaattgt aatgtatgca gaaaatgtgg acctggaatg tccagatact 19140 catgttgtgt acaaacctgg agtttcagat gctagttctg agaccaatct tggacaacag 19200 tctatgccca acagacccaa ctacattggc ttcagagata acttcatcgg acttatgtac 19260 tataacagta ctggcaacat gggggtactg gctggccaag cgtctcagtt gaatgcagtg 19320 gttgacttgc aggacagaaa cacagaactg tcttaccaac tcttgcttga ctctctgggc 19380 gacagaacca gatactttag catgtggaat caggctgtgg acagttatga tcctgatgta 19440 cgtgttattg aaaatcatgg tgtggaagat gaacttccca actattgttt tccgttggat 19500 ggtgtcggtc cgcgaacaga tagttacaag gagattaagc caaatggaga ccaatctact 19560 tggacaaatg tagacccaac tggcagcagt gaacttgcta agggaaatcc atttgccatg 19620 gaaattaacc ttcaagccaa tctatggcga agtttccttt attccaatgt ggctctatat 19680 ctcccagact cgtacaaata caccccgtcc aatgtcactc ttccagaaaa caaaaacacc 19740 tacgactaca tgaacgggcg ggtggtgccg ccatctctag tagacaccta tgtgaacatt 19800 ggtgccaggt ggtctctgga tgccatggac aatgtcaacc cattcaacca ccaccgtaac 19860 gctggcttgc gttaccgatc catgcttctg ggtaacggac gttatgtgcc tttccacata 19920 caagtgcctc aaaaattctt cgctgttaaa aacctgctgc ttctcccagg ctcctacact 19980 tatgagtgga actttaggaa ggatgtaaac atggttctac agagttccct cggtaacgac 20040 ctacgggtag atggcgccag catcagtttt acgagcatca acctctatgc tacttttttc 20100 cccatggctc acaacaccgc ttccaccctt gaagccatgc tgcggaatga caccaatgat 20160 cagtcattca acgactacct atctgcagct aacatgctct accccattcc tgccaatgca 20220 accaatattc ccatttccat tccttctcgc aactgggcgg ctttcagagg ctggtcattt 20280 accagactga aaaccaaaga aactccctct ttggggtctg gatttgaccc ctacttcgtc 20340 tattctggtt ctattcccta cctggatggt accttctacc tgaaccacac ttttaagaag 20400 gtttccatca tgtttgactc ttcagtgagc tggcctggaa atgacaggtt actatctcct 20460 aacgaatttg aaataaagcg cactgtggat ggcgaaggct acaacgtagc ccaatgcaac 20520 atgaccaaag actggttctt ggtacagatg ctcgccaact acaacatcgg ctatcagggc 20580 ttctacattc cagaaggata caaagatcgc atgtattcat ttttcagaaa cttccagccc 20640 atgagcaggc aggtggttga tgaggtcaat tacaaagact tcaaggccgt cgccataccc 20700 taccaacaca acaactctgg ctttgtgggt tacatggctc cgaccatgcg tcaaggtcaa 20760 ccctatcccg ctaactatcc ctatccactc attggaacaa ctgccgtaaa tagtgttacg 20820 cagaaaaagt tcttgtgtga cagaaccatg tggcgcatac cgttctcaag caacttcatg 20880 tctatgggag cccttacaga cttgggacag aacatgctct atgccaactc agctcatgct 20940 ctggacatga cctttgaggt ggatcccatg gatgagccca ccctgcttta tcttctcttc 21000 gaagttttcg acgtggtcag agtgcatcag ccacaccgcg gcatcatcga ggcagtctac 21060 ctgcgtacac cgttctcggc cggtaacgct accacgtaag aagcttcttg cttcttgcaa 21120 acagcagctg caaccatggc ctgcggatcc caaaacggct ccagcgagca agagctcaga 21180 gccattgtcc aagacctggg ttgcggacca tattttttgg gaacctttga taagcgcttc 21240 ccggggttca tggcccccga taagctcgcc tgtgccattg taaatacggc cggacgtgag 21300 acggggggag agcactggtt ggctttcggt tggaacccac gttctaacac ctgctacctt 21360 tttgatcctt ttggattctc ggatgatcgt ctcaaacaga tttaccagtt tgaatatgag 21420 ggtctcctgc gccgcagcgc tcttgctacc aaggaccggt gtattacgct ggaaaaatct 21480 acccagaccg tgcagggccc ccgttctgcc gcctgcggac ttttctgctg catgttcctt 21540 catgcctttg tgcactggcc tgaccgtccc atggacggaa accccaccat gaaattgcta 21600 actggagtgc caaacaacat gcttcattct cctaaagtcc agcccaccct gtgtgacaat 21660 caaaaagcac tctaccattt tctcaatacc cattcgcctt attttcgctc tcatcgtaca 21720 cacatcgaaa gggccactgc gttcgaccgt atggatgtgc aataatgatt catgtaaaca 21780 acgtgttcaa taaacagcac tttatttttt acatgtatcg aggctctgga ttacttattt 21840 atttacaagt cgaatgggtt ctgacgagaa tcagaatgac ccgcaggcag tgatacgttg 21900 cggaactgat acttgggttg ccacttgaat tcgggaatca ccaacttggg aaccggtata 21960 tcgggcagga tgtcactcca cagctttctg gtcagctgca aagctcccag caggtcagga 22020 gccgaaatct tgaaatcaca attaggacca gtgctctgag cgcgagagtt gcggtacacc 22080 ggattgcagc actgaaacac catcagcgac ggatgtctta cgcttgccag cacggtggga 22140 tctgcaatca tgcccacatc cagatcttca gcattggcaa tgctgaacgg ggtcatcttg 22200 caggtctgcc tacccatggc gggcacccaa ttaggcttgt ggttacaatc gcagtgcagg 22260 gggatcagta tcatcttggc ctgatcctgt ctgattcctg gatacacggc tctcatgaaa 22320 gcatcatatt gcttgaaagc ctgctgggct ttactaccct cggtataaaa catcccgcag 22380 gacctgctcg aaaactggtt agctgcgcag ccggcatcat tcacacagca gcgggcgtca 22440 ttgttggcta tttgcaccac acttctgccc cagcggtttt gggtgatttt ggttcgctcg 22500 ggattctcct tcaaggctcg ttgtccgttc tcgctggcca catccatctc gataatctgc 22560 tccttctgaa tcataatatt gccatgcaag cacttcagct tgccctcata atcattgcag 22620 ccatgaggcc acaacgcaca gcctgtacat tcccaattat ggtgggcgat ctgagaaaaa 22680 gaatgtatca ttccctgcag aaatcttccc atcatcgtgc tcagtgtctt gtgactagtg 22740 aaagttaact ggatgcctcg gtgctcctcg ttcacgtact ggtgacagat gcgcttgtat 22800 tgttcgtgct gctcaggcat tagtttaaaa gaggttctaa gttcgttatc cagcctgtac 22860 ttctccatca gcagacacat cacttccatg cctttctccc aagcagacac caggggcaag 22920 ctaatcggat tcttaacagt gcaggcagca gctcctttag ccagagggtc atctttggcg 22980 atcttctcaa tgcttctttt gccatccttc tcaacgatgc gcacgggcgg gtagctgaaa 23040 cccactgcta caagttgcgc ctcttctctt tcttcttcgc tgtcttgact gatgtcttgc 23100 atggggacat gtttggtctt ccttggcttc tttttcgggg gtatcggagg aggaggactg 23160 tcgctccgtt ccggagacag ggaggattgt gacgtttcgc tcaccattac caactgactg 23220 tcggtagaag aacctgaccc cacacggcga caggtgtttc tcttcggggg cagaggtgga 23280 ggcgattgcg aagggctgcg gtccgacctg gaaggcggat gactggcaga accccttccg 23340 cgttcggggg tgtgctccct gtggcggtcg cttaactgat ttccttcgcg gctggccatt 23400 gtgttctcct aggcagagaa acaacagaca tggaaactca gccattgctg tcaacatcgc 23460 cacgagtgcc atcacatctc gtcctcagcg acgaggaaaa ggagcagagc ttaagcattc 23520 caccgcccag tcctgccacc acctctaccc tagaagataa ggaggtcgac gcatctcatg 23580 acatgcagaa taaaaaagcg aaagagtctg agccagacat cgaacaagac ccgggctatg 23640 tgacaccggt ggaacacgag gaagagttga aacgctttct agagagagag gatgaaaact 23700 gcccaaaaca gcaagcggat aactatcacc aagatgctgg aaatagggat cagaacaccg 23760 actacctcat agggcttgac ggggaagacg cgctccttaa acatctagca agacagtcac 23820 tcatagtcaa ggatgcatta ttggacagaa ctgaagtgcc catcagtgtc gaagagctca 23880 gccgcgccta cgagcttaac ctattttcac ctcgtactcc ccccaaacgt cagccaaacg 23940 gcacctgcga gccaaatcct cgcttaaact tttatccagc ttttgctgtg ccagaagtac 24000 tggctaccta tcacatcttt tttaaaaatc aaaaaattcc agtctcctgc cgcgctaatc 24060 gcacccgcgc cgatgcccta ctcaatctgg gacctggttc acgcttacct gatatagctt 24120 ccttggaaga ggttccaaag atcttcgagg gtctgggcaa taatgagact cgggccgcaa 24180 atgctctgca aaagggagaa aatggcatgg atgagcatca cagcgttctg gtggaattgg 24240 aaggcgataa tgccagactc gcagtactca agcgaagcgt cgaggtcaca cactttgcat 24300 accccgctgt caacctgccc cctaaagtca tgacggccgt catggaccag ttactcatta 24360 agcgcgcaag tcccctttca gaagacatgc atgacccaga tgcctgtgat gagggtaaac 24420 cagtggtcag tgatgagcag ctaacccgat ggctgggcac cgactctccc cgggatttgg 24480 aagagcgtcg caagcttatg atggccgtgg tgctggttac cgtagaacta gagtgtcttc 24540 ggcgtttctt taccgattca gaaaccttgc gcaaactcga agagaatctg cactacactt 24600 ttagacacgg ctttgtgcgg caggcatgca agatatctaa cgtggaactc accaacctgg 24660 tttcctacat gggtattctg catgagaatc gcctaggaca aagcgtgctg cacagcaccc 24720 ttaaggggga agcccgccgt gattacatcc gcgattgtgt ttatctctac ctgtgccaca 24780 cgtggcaaac cggcatgggt gtatggcagc aatgtttaga agaacagaac ctgaaagagc 24840 taaacaagct cttacagaaa tctcttaagg ttctgtggac agggttcgac gagcgcaccg 24900 tcgcttccga cctggcagac ctcatcttcc cagagcgtct cagggttact ttgcgaaacg 24960 gactgcctga ctttatgagc cagagcatgc ttaacaattt tcgctctttc atcctggaac 25020 gctccggtat cctgcccgcc acctgctgcg cactgccctc cgactttgtg cctctcacct 25080 accgcgaatg ccccccgccg ctatggagtc actgctacct gttccgtctg gccaactacc 25140 tctcctacca ctcggatgtg atcgaggatg tgagcggaga cggcttgctg gagtgtcact 25200 gccgctgcaa tctgtgcacg ccccaccggt ccctagcttg caacccccag ttgatgagcg 25260 aaacccagat aataggcacc tttgaattgc aaggccccag cagccaaggc gatgggtctt 25320 ctcctgggca aagtttaaaa ctgaccccgg gactgtggac ctccgcctac ttgcgcaagt 25380 ttgccccgga agattaccac ccctatgaaa tcaagttcta tgaggaccaa tcacagcctc 25440 cgaaagccga actttcggcc tgcgtcatca cccagggggc aattctggcc caattgcaag 25500 ccatccaaaa atcccgccaa gaatttctac tgaaaaaggg taagggggtc taccttgacc 25560 cccagaccgg cgaggaactc aacacaaggt tccctcagga tgtcccaacg acgagaaagc 25620 aagaagttga aggtgcagcc gccgccccca gaagatatgg aggaagattg ggacagtcag 25680 gcagaggaag cggaggagga ggacagtctg gaggacagtc tggaggaaga cagtttggag 25740 gaggaaaacg aggaggcaga ggaggtggaa gaagtaaccg ccgacaaaca gttatcctcg 25800 gctgcggaga caagcaacag cgctaccatc tccgctccga gtcgaggaac ccggcggcgt 25860 cccagcagta gatgggacga gaccggacgc ttcccgaacc caaccagcgc ttccaagacc 25920 ggtaagaagg atcggcaggg atacaagtcc tggcgggggc ataagaatgc catcatctcc 25980 tgcttgcatg agtgcggggg caacatatcc ttcacgcggc gctacttgct attccaccat 26040 ggggtgaact ttccgcgcaa tgttttgcat tactaccgtc acctccacag cccctactat 26100 agccagcaaa tcccggcagt ctcgacagat aaagacagcg gcggcgacct ccaacagaaa 26160 accagcagcg gcagttagaa aatacacaac aagtgcagca acaggaggat taaagattac 26220 agccaacgag ccagcgcaaa cccgagagtt aagaaatcgg atctttccaa ccctgtatgc 26280 catcttccag cagagtcggg gccaagagca ggaactgaaa ataaaaaacc gatctctgcg 26340 ttcgctcacc agaagttgtt tgtatcacaa gagcgaagat caacttcagc gcactctcga 26400 ggacgccgag gctctcttca acaagtactg cgcgctgact cttaaagagt aggcagcgac 26460 cgcgcttatt caaaaaaggc gggaattaca tcatcctcga catgagtaaa gaaattccca 26520 cgccttacat gtggagttat cagccccaaa tgggattggc ggcaggcgcc tcccaggact 26580 actccacccg catgaattgg ctcagcgccg ggccttctat gatttctcga gttaatgata 26640 tacgcgccta ccgaaaccaa atacttttgg aacagtcagc tcttaccacc acgccccgcc 26700 aacaccttaa tcccagaaat tggcccgccg ccctagtgta ccaggaaagt cccgctccca 26760 ccactgtatt acttcctcga gacgcccagg ccgaagtcca aatgactaat gcaggtgcgc 26820 agttagcggg cggctccacc ctatgtcgtc acaggcctcg gcataatata aaacgcctga 26880 tgatcagagg ccgaggtatc cagctcaacg acgagtcggt gagctctccg cttggtctac 26940 gaccagacgg aatctttcag attgccggct gcgggagatc ttccttcacc cctcgtcagg 27000 ctgttctgac tttggaaagt tcgtcttcgc aaccccgctc gggcggaatc gggaccgttc 27060 aatttgtgga ggagtttact ccctctgtct acttcaaccc cttctccgga tctcctgggc 27120 actacccgga cgagttcata ccgaacttcg acgcgattag cgagtcagtg gacggctacg 27180 attgatgtct ggtgacgcgg ctgagctatc tcggctgcga catctagacc actgccgccg 27240 ctttcgctgc tttgcccggg aactcattga gttcatctac ttcgaactcc ccaaggatca 27300 ccctcaaggt ccggcccacg gagtgcggat tactatcgaa ggcaaaatac actctcgcct 27360 gcaacgaatt ttctcccagc ggcccgtgct gatcgagcga gaccagggaa acaccacggt 27420 ttccatctac tgcatttgta atcaccccgg attgcatgaa agcctttgct gtcttatgtg 27480 tactgagttt aataaaaact gaattaagac tctcctacgg actgccgctt cttcaacccg 27540 gattttacaa ccagaagaac gaaacttttc ctgtcgtcca ggactctgtt aacttcacct 27600 ttcctactca caaactagaa gctcaacgac tacaccgctt ttccagaagc attttcccta 27660 ctaatactac tttcaaaacc ggaggtgagc tccaaggtct tcctacagaa aacccttggg 27720 tggaagcggg ccttgtagtg ctaggaattc ttgcgggtgg gcttgtgatt attctttgct 27780 acctatacac accttgcttc actttcctag tggtgttgtg gtattggttt aaaaaatggg 27840 gcccatacta gtcttgcttg ttttactttc gcttttggaa ccgggttctg ccaattacga 27900 tccatgtcta gacttcgacc cagaaaactg cacacttact tttgcacccg acacaagccg 27960 catctgtgga gttcttatta agtgcggatg ggaatgcagg tccgttgaaa ttacacacaa 28020 taacaaaacc tggaacaata ccttatccac cacatgggag ccaggagttc ccgagtggta 28080 cactgtctct gtccgaggtc ctgacggttc catccgcatt agtaacaaca ctttcatttt 28140 ttctgaaatg tgcgatctgg ccatgttcat gagcaaacag tattctctat ggcctcctag 28200 caaggacaac atcgtaacgt tctccattgc ttattgcttg tgcgcttgcc ttcttactgc 28260 tttactgtgc gtatgcatac acctgcttgt aaccactcgc atcaaaaacg ccaataacaa 28320 agaaaaaatg ccttaacctc tttctgttta cagacatggc ttctcttaca tctctcatat 28380 ttgtcagcat tgtcactgcc gctcacggac aaacagtcgt ctctatccct ctaggacata 28440 attacactct cataggaccc ccaatcactt cagaggtcat ctggaccaaa ctgggaagcg 28500 ttgattactt tgatataatc tgcaacaaaa caaaaccaat aatagtaact tgcaacatac 28560 aaaatcttac attgattaat gttagcaaag tttacagcgg ttactattat ggttatgaca 28620 gatacagtag tcaatataga aattacttgg ttcgtgttac ccagttaaaa accacgaaaa 28680 tgccaaatat ggcaaagatt cgatccgatg acaattctct agaaactttt acatctccca 28740 ccacacccga cgaaaaaaac atcccagatt caatgattgc aattgttgca gcggtggcag 28800 tggtgatggc actaataata atatgcatgc ttttatatgc ttgtcgctac aaaaagtttc 28860 atcctaaaaa acaagatctc ctactaaggc ttaacattta atttcttttt atacagccat 28920 ggtttccact accacattcc ttatgcttac tagtcttgca actctgactt ctgctcgctc 28980 acacctcact gtaactatag gctcaaactg cacactaaaa ggacctcaag gtggtcatgt 29040 cttttggtgg agaatatatg acaatggatg gtttacaaaa ccatgtgacc aacctggtag 29100 atttttctgc aacggcagag acctaaccat tatcaacgtg acagcaaatg acaaaggctt 29160 ctattatgga accgactata aaagtagttt agattataac attattgtac tgccatctac 29220 cactccagca ccccgcacaa ctactttctc tagcagcagt gtcgctaaca atacaatttc 29280 caatccaacc tttgccgcgc ttttaaaacg cactgtgaat aattctacaa cttcacatac 29340 aacaatttcc acttcaacaa tcagcattat cgctgcagtg acaattggaa tatctattct 29400 tgtttttacc ataacctact acgcctgctg ctatagaaaa gacaaacata aaggtgatcc 29460 attacttaga tttgatattt aatttgttct tttttttttt atttacagta tggtgaacac 29520 caatcatggt acctagaaat ttcttcttca ccatactcat ttgtgcattt aatgtttgcg 29580 ctactttcac agcagtagcc acagcaaccc cagactgtat aggagcattt gcttcctatg 29640 cactttttgc ttttgttact tgcatctgcg tatgtagcat agtctgcctg gttattaatt 29700 ttttccaact tctagactgg atccttgtgc gaattgccta cctgcgccac catcccgaat 29760 accgcaacca aaatatcgcg gcacttctta gactcatcta aaaccatgca ggctatacta 29820 ccaatatttt tgcttctatt gcttccctac gctgtctcaa ccccagctgc ctatagtact 29880 ccaccagaac accttagaaa atgcaaattc caacaaccgt ggtcatttct tgcttgctat 29940 cgagaaaaat cagaaattcc cccaaattta ataatgattg ctggaataat taatataatc 30000 tgttgcacca taatttcatt tttgatatac cccctatttg attttggctg gaatgctccc 30060 aatgcacatg atcatccaca agacccagag gaacacattc ccctacaaaa catgcaacat 30120 ccaatagcgc taatagatta cgaaagtgaa ccacaacccc cactactccc tgctattagt 30180 tacttcaacc taaccggcgg agatgactga aacactcacc acctccaatt ccgccgagga 30240 tctgctcgat atggacggcc gcgtctcaga acagcgactt gcccaactac gcatccgcca 30300 gcagcaggaa cgcgcggcca aagagctcag agatgtcatc caaattcacc aatgcaaaaa 30360 aggcatattc tgtttggtaa aacaagccaa gatatcctac gagatcaccg ctactgacca 30420 tcgcctctct tacgaacttg gcccccaacg acaaaaattt acctgcatgg tgggaatcaa 30480 ccccatagtt atcacccagc aaagtggaga tactaagggt tgcattcact gctcctgcga 30540 ttccatcgag tgcacctaca ccctgctgaa gaccctatgc ggcctaagag acctgctacc 30600 aatgaattaa aaaatgatta ataaaaaatc acttacttga aatcagcaat aaggtctctg 30660 ttgaaatttt ctcccagcag cacctcactt ccctcttccc aactctggta ttctaaaccc 30720 cgttcagcgg catactttct ccatacttta aaggggatgt caaattttag ctcctctcct 30780 gtacccacaa tcttcatgtc tttcttccca gatgaccaag agagtccggc tcagtgactc 30840 cttcaaccct gtctacccct atgaagatga aagcacctcc caacacccct ttataaaccc 30900 agggtttatt tccccaaatg gcttcacaca aagcccagac ggagttctta ctttaaaatg 30960 tttaacccca ctaacaacca caggcggatc tctacagcta aaagtgggag ggggacttac 31020 agtggatgac actgatggta ccttacaaga aaacatacgt gctacagcac ccattactaa 31080 aaataatcac tctgtagaac tatccattgg aaatggatta gaaactcaaa acaataaact 31140 atgtgccaaa ttgggaaatg ggttaaaatt taacaacggt gacatttgta taaaggatag 31200 tattaacacc ttatggactg gaataaaccc tccacctaac tgtcaaattg tggaaaacac 31260 taatacaaat gatggcaaac ttactttagt attagtaaaa aacggagggc ttgttaatgg 31320 ctacgtgtct ctagttggtg tatcagacac tgtgaaccaa atgttcacac aaaagacagc 31380 aaacatccaa ttaagattat attttgactc ttctggaaat ctattaactg atgaatcaga 31440 cttaaaaatt ccacttaaaa ataaatcttc tacagcgacc agtgaaactg tagccagcag 31500 caaagccttt atgccaagta ctacagctta tcccttcaac accactacta gggatagtga 31560 aaactacatt catggaatat gttactacat gactagttat gatagaagtc tatttccctt 31620 gaacatttct ataatgctaa acagccgtat gatttcttcc aatgttgcct atgccataca 31680 atttgaatgg aatctaaatg caagtgaatc tccagaaagc aacatagcta cgctgaccac 31740 atcccccttt ttcttttctt acattacaga agacgacaac taaaataaag tttaagtgtt 31800 tttatttaaa atcacaaaat tcgagtagtt attttgcctc caccttccca tttgacagaa 31860 tacaccaatc tctccccacg cacagcttta aacatttgga taccattaga gatagacatt 31920 gttttagatt ccacattcca aacagtttca gagcgagcca atctggggtc agtgatagat 31980 aaaaatccat cgcgatagtc ttttaaagcg ctttcacagt ccaactgctg cggatgcgaa 32040 tccggagtct ggatcacggt catctggaag aagaacgatg ggaatcataa tccgaaaacg 32100 gtatcggacg attgtgtctc atcaaaccca caagcagccg ctgtctgcgt cgctccgtgc 32160 aactgctgtt tatgggatca gggtccacag tgtcctgaag catgatttta atagccctta 32220 acatcaactt tctggtgcga tgcgcgcagc aacgcattct gatttcactc aaatctttgc 32280 agtaggtaca acacattatt acaatattgt ttaataaacc ataattaaaa gcgctccagc 32340 caaaactcat atctgatata atcgcccctg catgaccatc ataccaaagt ttaatataaa 32400 ttaaatgacg ttccctcaaa aacacactac ccacatacat gatctctttt ggcatgtgca 32460 tattaacaat ctgtctgtac catggacaac gttggttaat catgcaaccc aatataacct 32520 tccggaacca cactgccaac accgctcccc cagccatgca ttgaagtgaa ccctgctgat 32580 tacaatgaca atgaagaacc caattctctc gaccgtgaat cacttgagaa tgaaaaatat 32640 ctatagtggc acaacataga cataaatgca tgcatcttct cataattttt aactcctcag 32700 gatttagaaa catatcccag ggaataggaa gctcttgcag aacagtaaag ctggcagaac 32760 aaggaagacc acgaacacaa cttacactat gcatagtcat agtatcacaa tctggcaaca 32820 gcgggtggtc ttcagtcata gaagctcggg tttcattttc ctcacaacgt ggtaactggg 32880 ctctggtgta agggtgatgt ctggcgcatg atgtcgagcg tgcgcgcaac cttgtcataa 32940 tggagttgct tcctgacatt ctcgtatttt gtatagcaaa acgcggccct ggcagaacac 33000 actcttcttc gccttctatc ctgccgctta gcgtgttccg tgtgatagtt caagtacagc 33060 cacactctta agttggtcaa aagaatgctg gcttcagttg taatcaaaac tccatcgcat 33120 ctaattgttc tgaggaaatc atccacggta gcatatgcaa atcccaacca agcaatgcaa 33180 ctggattgcg tttcaagcag gagaggagag ggaagagacg gaagaaccat gttaattttt 33240 attccaaacg atctcgcagt acttcaaatt gtagatcgcg cagatggcat ctctcgcccc 33300 cactgtgttg gtgaaaaagc acagctaaat caaaagaaat gcgattttca aggtgctcaa 33360 cggtggcttc caacaaagcc tccacgcgca catccaagaa caaaagaata ccaaaagaag 33420 gagcattttc taactcctca atcatcatat tacattcctg caccattccc agataatttt 33480 cagctttcca gccttgaatt attcgtgtca gttcttgtgg taaatccaat ccacacatta 33540 caaacaggtc ccggagggcg ccctccacca ccattcttaa acacaccctc ataatgacaa 33600 aatatcttgc tcctgtgtca cctgtagcga attgagaatg gcaacatcaa ttgacatgcc 33660 cttggctcta agttcttctt taagttctag ttgtaaaaac tctctcatat tatcaccaaa 33720 ctgcttagcc agaagccccc cgggaacaag agcaggggac gctacagtgc agtacaagcg 33780 cagacctccc caattggctc cagcaaaaac aagattggaa taagcatatt gggaaccgcc 33840 agtaatatca tcgaagttgc tggaaatata atcaggcaga gtttcttgta aaaattgaat 33900 aaaagaaaaa tttgccaaaa aaacattcaa aacctctggg atgcaaatgc aataggttac 33960 cgcgctgcgc tccaacattg ttagttttga attagtctgc aaaaataaaa aaaaaaacaa 34020 gcgtcatatc atagtagcct gacgaacagg tggataaatc agtctttcca tcacaagaca 34080 agccacaggg tctccagctc gaccctcgta aaacctgtca tggtgattaa acaacagcac 34140 cgaaagttcc tcgcggtgac cagcatgaat aattcttgat gaagcataca atccagacat 34200 gttagcatca gttaacgaga aaaaacagcc aacatagcct ttgggtataa ttatgcttaa 34260 tcgtaagtat agcaaagcca cccctcgcgg atacaaagta aaaggcacag gagaataaaa 34320 aatataatta tttctctgct gctgttcagg caacgtcgcc cccggtccct ctaaatacac 34380 atacaaagcc tcatcagcca tggcttacca gacaaagtac agcgggcacg cacaagctct 34440 aaagtcactc tccaacctct ccacaatata tatacacaag ccctaaactg acgtaatggg 34500 agtaaagtgt aaaaaatccc gccaaaccca acacacaccc cgaaactgcg tcaccaggga 34560 aaagtacagt ttcacttccg caatcccaac aagcgtcact tcctctttct cacggtacgt 34620 cacatcccat taacttgcaa cgtcattttc ccacggccgc gccgccccgt ttagccgtta 34680 accccacagc caatcaccac acaccccaca atttttaaaa tcacctcatt tacatattgg 34740 caccattcca tctataaggt atattattga tgatg 34775 

What is claimed is:
 1. A means for propagating replication-defective adenovirus in an adenoviral E1-complementing cell line expressing E1 gene product(s) which are non-native to the adenovirus, which comprises: (a) inserting all or a portion of a heterologous adenoviral E4 region comprising nucleic acid sequence encoding open reading frame 6 (ORF6) into a replication-defective adenovirus; wherein the E4 region or portion thereof inserted into the adenovirus is native to a virus of the same adenovirus serotype as the E1 gene product(s) expressed by the complementing cell line; (b) introducing the replication-defective adenovirus into the adenoviral E1-complementing cell line; (c) allowing the replication-defective adenovirus to propagate in the adenoviral E1-complementing cell line; and (d) rescuing the propagated adenovirus.
 2. A means in accordance with claim 1 wherein the heterologous adenoviral E4 region or portion thereof comprises the complete adenoviral E4-encoding region.
 3. A means in accordance with claim 2 wherein the heterologous adenoviral E4 region or portion thereof comprises the complete adenoviral E4-encoding region and native E4 promoter.
 4. A means in accordance with claim 1 wherein the heterologous adenoviral E4 region or portion thereof is inserted into the replication-defective virus in place of nucleic acid sequence encoding open reading frame 6 (ORF6).
 5. A means in accordance with claim 1 wherein the heterologous adenoviral E4 region or portion thereof is inserted into the replication-defective virus in place of nucleic acid sequence encoding the complete adenoviral E4-encoding region.
 6. A means in accordance with claim 1 wherein the heterologous adenoviral E4 region or portion thereof is derived from a subgroup C adenovirus.
 7. A means in accordance with claim 1 wherein the subgroup C adenovirus is adenovirus of serotype
 5. 8. A means in accordance with claim 7 wherein the replication-defective adenovirus is an adenovirus of subgroup B.
 9. A means in accordance with claim 7 wherein the replication-defective adenovirus is an adenovirus of serotype
 35. 10. A means in accordance with claim 1 wherein the heterologous adenoviral E4 region or portion thereof is operatively linked to a heterologous promoter.
 11. A means in accordance with claim 1 wherein the adenoviral E1-complementing cell line is a PER.C6® cell line.
 12. A replication-defective adenovirus comprising all or a portion of a heterologous E4 region comprising a heterologous adenoviral open reading frame 6 (ORF6).
 13. A replication-defective adenovirus in accordance with claim 12 wherein the adenovirus comprises a heterologous gene of interest.
 14. A replication-defective adenovirus in accordance with claim 13 wherein the heterologous gene of interest is a gene encoding an HIV-1 antigen.
 15. A replication-defective adenovirus in accordance with claim 14 wherein the HIV-1 antigen is selected from the group consisting of HIV-1 gag, pol, nef and env.
 16. A replication-defective adenovirus comprising all or a portion of a heterologous E4 region comprising a heterologous adenoviral open reading frame 6 (ORF6) and a gene encoding HIV-1 gag.
 17. A replication-defective adenovirus comprising all or a portion of a heterologous E4 region comprising a heterologous adenoviral open reading frame 6 (ORF6) in place of a native E4 region or portion thereof comprising ORF6.
 18. A replication-defective adenovirus comprising all or a portion of a heterologous E4 region comprising a complete heterologous E4 region in place of a complete native E4 region.
 19. A replication-defective adenovirus comprising a heterologous E4 region or portion thereof comprising a complete heterologous E4 region including E4 promoter in place of a complete native E4 region.
 20. Adenovirus propagated in accordance with the means of claim
 1. 21. A means in accordance with claim 1 wherein the replication-defective adenovirus comprises a heterologous gene of interest.
 22. A means in accordance with claim 21 wherein the heterologous gene of interest is a gene encoding an HIV-1 antigen.
 23. A means in accordance with claim 22 wherein the HIV-1 antigen is selected from the group consisting of: HIV-1 gag, pol, nef and env.
 24. A replication-defective adenovirus of serotype 35 comprising all or a portion of an adenovirus serotype 5 E4 region comprising open reading frame 6 (ORF6) and a heterologous gene of interest.
 25. A replication-defective adenovirus in accordance with claim 24 wherein the heterologous gene of interest is a gene encoding an HIV-1 antigen.
 26. A replication-defective adenovirus in accordance with claim 25 wherein the HIV-1 antigen is selected from the group consisting of: HIV-1 gag, pol, nef and env.
 27. A replication-defective adenovirus of serotype 35 comprising all or a portion of an adenovirus serotype 5 E4 region comprising open reading frame 6 (ORF6) and a gene encoding HIV-1 gag.
 28. A recombinant adenoviral vector of serotype 24 which comprises an E4 gene or a segment of an E4 gene comprising open reading frame 6 (“ORF6”) of an alternative serotype.
 29. A population of cells comprising the recombinant adenoviral vector of claim
 28. 30. A method for producing recombinant, replication-defective adenovirus particles comprising: (a) introducing a recombinant adenoviral vector of claim 28 into a population of cells expressing adenovirus E1; and (b) harvesting the resultant recombinant, replication-defective adenovirus.
 31. Purified recombinant, replication-defective adenovirus particles harvested in accordance with the method of claim
 30. 32. A composition comprising purified recombinant adenovirus particles in accordance with claim
 31. 33. A composition in accordance with claim 32 which comprises a physiologically acceptable carrier.
 34. A recombinant adenoviral vector in accordance with claim 28 which is at least partially deleted in E1 and devoid of E1 activity and comprises a heterologous nucleic acid.
 35. A composition comprising purified recombinant adenoviral particles in accordance with claim 31 which are at least partially deleted in E1 and devoid of E1 activity and comprise a heterologous nucleic acid.
 36. A method for effecting the delivery and expression of heterologous nucleic acid comprising administering the composition of claim 35 prior or subsequent to administration of the heterologous nucleic acid with the same or different vector.
 37. A method in accordance with claim 36 wherein the composition is preceded or followed by administration of heterologous nucleic acid with an adenovirus of a different serotype.
 38. A composition in accordance with claim 35 wherein the heterologous nucleic acid encodes an HIV antigen.
 39. A method for generating a cellular-mediated immune response against HIV in an individual comprising administering to the individual a composition of claim
 38. 40. A composition in accordance with claim 39 wherein the HIV antigen is HIV-1 gag or immunologically relevant modification thereof.
 41. A composition in accordance with claim 39 wherein the HIV antigen is HIV-1 nef or immunologically relevant modification thereof.
 42. A composition in accordance with claim 39 wherein the HIV antigen is HIV-1 pol or immunologically relevant modification thereof.
 43. A recombinant adenoviral vector of serotype 24 which is at least partially deleted in E1 and devoid of E1 activity; wherein said vector comprises an E4 gene or a segment of an E4 gene from adenovirus serotype 5 comprising open reading frame 6 (“ORF6”), and a heterologous nucleic acid.
 44. A population of cells comprising the recombinant adenoviral vector of claim
 43. 45. A method for producing recombinant, replication-defective adenovirus particles comprising: (a) introducing a recombinant adenoviral vector of claim 43 into a population of cells expressing adenovirus serotype 5 E1; and (b) harvesting the resultant recombinant, replication-defective adenovirus.
 46. Purified recombinant, replication-defective adenovirus particles harvested in accordance with the method of claim
 45. 47. A composition comprising purified recombinant adenovirus particles in accordance with claim
 46. 48. A composition in accordance with claim 47 which comprises a physiologically acceptable carrier.
 49. A method for effecting the delivery and expression of the heterologous nucleic acid comprising administering the composition of claim 48 prior or subsequent to administration of the heterologous nucleic acid with the same or different vector.
 50. A method in accordance with claim 49 above wherein the composition is preceded or followed by administration of the heterologous nucleic acid with an adenovirus of a different serotype.
 51. A composition in accordance with claim 48 wherein the heterologous nucleic acid encodes an HIV antigen.
 52. A method for generating a cellular-mediated immune response against HIV in an individual comprising administering to the individual a composition of claim
 51. 53. A composition in accordance with claim 51 wherein the HIV antigen is HIV-1 gag or immunologically relevant modification thereof.
 54. A composition in accordance with claim 51 wherein the HIV antigen is HIV-1 nef or immunologically relevant modification thereof.
 55. A composition in accordance with claim 51 wherein the HIV antigen is HIV-1 pol or immunologically relevant modification thereof. 56 A recombinant adenoviral vector of serotype 34 which comprises an E4 gene or a segment of an E4 gene comprising open reading frame 6 (“ORF6”) of an alternative serotype.
 57. A population of cells comprising the recombinant adenoviral vector of claim
 56. 58. A method for producing recombinant, replication-defective adenovirus particles comprising: (a) introducing a recombinant adenoviral vector of claim 56 into a population of cells expressing adenovirus E1; and (b) harvesting the resultant recombinant, replication-defective adenovirus.
 59. Purified recombinant, replication-defective adenovirus particles harvested in accordance with the method of claim
 58. 60. A composition comprising purified recombinant adenovirus particles in accordance with claim
 59. 61. A composition in accordance with claim 60 which comprises a physiologically acceptable carrier.
 62. A recombinant adenoviral vector in accordance with claim 56 which is at least partially deleted in E1 and devoid of E1 activity and comprises a heterologous nucleic acid.
 63. A composition comprising purified recombinant adenoviral particles in accordance with claim 59 which are at least partially deleted in E1 and devoid of E1 activity and comprise a heterologous nucleic acid.
 64. A method for effecting the delivery and expression of heterologous nucleic acid comprising administering the composition of claim 63 prior or subsequent to administration of the heterologous nucleic acid with the same or different vector.
 65. A method in accordance with claim 64 wherein the composition is preceded or followed by administration of heterologous nucleic acid with an adenovirus of a different serotype.
 66. A composition in accordance with claim 63 wherein the heterologous nucleic acid encodes an HIV antigen.
 67. A method for generating a cellular-mediated immune response against HIV in an individual comprising administering to the individual a composition of claim
 66. 68. A composition in accordance with claim 67 wherein the HIV antigen is HIV-1 gag or immunologically relevant modification thereof.
 69. A composition in accordance with claim 67 wherein the HIV antigen is HIV-1 nef or immunologically relevant modification thereof.
 70. A composition in accordance with claim 67 wherein the HIV antigen is HIV-1 pol or immunologically relevant modification thereof.
 71. A recombinant adenoviral vector of serotype 34 which is at least partially deleted in E1 and devoid of E1 activity; wherein said vector comprises an E4 gene or a segment of an E4 gene from adenovirus serotype 5 comprising open reading frame 6 (“ORF6”), and a heterologous nucleic acid.
 72. A population of cells comprising the recombinant adenoviral vector of claim
 71. 73. A method for producing recombinant, replication-defective adenovirus particles comprising: (a) introducing a recombinant adenoviral vector of claim 71 into a population of cells expressing adenovirus serotype 5 E1; and (b) harvesting the resultant recombinant, replication-defective adenovirus.
 74. Purified recombinant, replication-defective adenovirus particles harvested in accordance with the method of claim
 73. 75. A composition comprising purified recombinant adenovirus particles in accordance with claim
 74. 76. A composition in accordance with claim 75 which comprises a physiologically acceptable carrier.
 77. A method for effecting the delivery and expression of the heterologous nucleic acid comprising administering the composition of claim 76 prior or subsequent to administration of the heterologous nucleic acid with the same or different vector.
 78. A method in accordance with claim 77 above wherein the composition is preceded or followed by administration of the heterologous nucleic acid with an adenovirus of a different serotype.
 79. A composition in accordance with claim 76 wherein the heterologous nucleic acid encodes an HIV antigen.
 80. A method for generating a cellular-mediated immune response against HIV in an individual comprising administering to the individual a composition of claim
 79. 81. A composition in accordance with claim 79 wherein the HIV antigen is HIV-1 gag or immunologically relevant modification thereof.
 82. A composition in accordance with claim 79 wherein the HIV antigen is HIV-1 nef or immunologically relevant modification thereof.
 83. A composition in accordance with claim 79 wherein the HIV antigen is HIV-1 pol or immunologically relevant modification thereof. 